U.S. patent application number 13/302654 was filed with the patent office on 2012-04-05 for control of atp release by red blood cells and therapeutic applications thereof.
This patent application is currently assigned to SAINT LOUIS UNIVERSITY. Invention is credited to Randy S. Sprague, Madelyn Stumpf.
Application Number | 20120083443 13/302654 |
Document ID | / |
Family ID | 38620255 |
Filed Date | 2012-04-05 |
United States Patent
Application |
20120083443 |
Kind Code |
A1 |
Sprague; Randy S. ; et
al. |
April 5, 2012 |
Control of ATP Release by Red Blood Cells and Therapeutic
Applications Thereof
Abstract
The invention is based upon the discovery that red blood cells
contain phosphodiesterase 3B (PDE3B), and that inhibition of that
phosphodiesterase allows for an enhanced accumulation of cAMP and
subsequent release of ATP. It was further discovered that RBCS
treated with insulin accumulate significantly less cAMP and release
significantly less ATP than normal RBCS. Likewise, RBCS of patients
suffering from type 2 diabetes (hyperinsulinemia) accumulate
significantly less cAMP and release significantly less ATP than
normal RBCS. It was further discovered that prostaglandin analogues
synergistically work with phosphodiesterase 3B inhibitors to
improve or increase cAMP accumulation and ATP release RBCS. Thus
the invention is directed to compositions and methods for improving
ATP release by RBCS, via administering PDE3B inhibitor or a
combination of PDE3B inhibitor and prostaglandin analogue.
Inventors: |
Sprague; Randy S.; (St.
Louis, MO) ; Stumpf; Madelyn; (St. Louis,
MO) |
Assignee: |
SAINT LOUIS UNIVERSITY
ST. LOUIS
MO
|
Family ID: |
38620255 |
Appl. No.: |
13/302654 |
Filed: |
November 22, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11695102 |
Apr 2, 2007 |
8084221 |
|
|
13302654 |
|
|
|
|
60788584 |
Apr 1, 2006 |
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Current U.S.
Class: |
514/6.5 ;
514/252.06; 514/263.36; 514/312; 514/332; 514/392 |
Current CPC
Class: |
A61K 31/557 20130101;
A61P 3/00 20180101; A61K 31/4709 20130101; A61P 11/00 20180101;
A61K 45/06 20130101; A61P 9/12 20180101; A61P 3/10 20180101; A61P
3/08 20180101; A61K 31/4709 20130101; A61K 2300/00 20130101; A61K
31/557 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/6.5 ;
514/332; 514/312; 514/392; 514/263.36; 514/252.06 |
International
Class: |
A61K 38/28 20060101
A61K038/28; A61K 31/4709 20060101 A61K031/4709; A61K 31/4166
20060101 A61K031/4166; A61K 31/522 20060101 A61K031/522; A61P 3/08
20060101 A61P003/08; A61P 3/10 20060101 A61P003/10; A61P 3/00
20060101 A61P003/00; A61P 9/12 20060101 A61P009/12; A61P 11/00
20060101 A61P011/00; A61K 31/444 20060101 A61K031/444; A61K 31/501
20060101 A61K031/501 |
Claims
1. A therapeutic composition comprising: a phosphodiesterase
inhibitor, wherein the phosphodiesterase inhibitor is a
phosphodiesterase 3 inhibitor; a prostaglandin, wherein the
prostaglandin is selected from the group consisting of iloprost,
UT-15C, and prostaglandin I.sub.2; and a pharmaceutically
acceptable excipient.
2. The therapeutic composition of claim 1, wherein the
phosphodiesterase 3 inhibitor is selected from the group consisting
of amrinone, cilostazol, CI-930, enoximone, meribendan, milrinone,
pentoxifylline, pimobendane, and 3-isobutyl-1-methylxanthine.
3. The therapeutic composition of claim 1, further comprising a
beta-adrenergic receptor agonist.
4. The therapeutic composition of claim 2, wherein the
beta-andrenergic agonist is selected from the group consisting of
epinephrine and isoproterenol.
5. A method of treating a disease or a condition, the method
comprising: administering a therapeutically effective amount of a
phosphodiesterase inhibitor to a subject, wherein the disease or
the condition is selected from the group consisting of pulmonary
hypertension, cystic fibrosis, hyperinsulinemia, prediabetes,
metabolic syndrome, type 1 diabetes, and type 2 diabetes.
6. The method of claim 5, wherein the phosphodiesterase inhibitor
is a phosphodiesterase 3 inhibitor.
7. The method of claim 5, wherein the phosphodiesterase inhibitor
is selected from the group consisting of amrinone, cilostazol,
CI-930, enoximone, meribendan, milrinone, pentoxifylline,
pimobendane, and 3-isobutyl-1-methylxanthine.
8. The method of claim 5, wherein blood flow is increased.
9. The method of claim 5 further comprising administering at least
one of a prostaglandin and a beta-adrenergic receptor agonist.
10. The method of claim 9, wherein the prostaglandin is selected
from the group consisting of iloprost, UT-15C, and prostaglandin
I.sub.2.
11. The method of claim 9, wherein the beta-adrenergic receptor
agonist is selected from the group consisting of epinephrine and
isoproterenol.
12. The method of claim 5, further comprising co-administration
with insulin.
13. A method of stimulating ATP release by a red blood cell in a
subject, the method comprising: administering to the subject a
therapeutically effective amount of a phosphodiesterase
inhibitor.
14. The method of claim 13, wherein the phosphodiesterase inhibitor
is a phosphodiesterase isoform 3 inhibitor.
15. The method of claim 14, wherein the phosphodiesterase inhibitor
is selected from the group consisting of amrinone, cilostazol,
CI-930, enoximone, meribendan, milrinone, pentoxifylline,
pimobendane, and 3-isobutyl-1-methylxanthine.
16. The method of claim 13, further comprising administering at
least one of a prostaglandin and a beta-adrenergic receptor
agonist.
17. The method of claim 16, wherein the prostaglandin is selected
from the group consisting of iloprost, UT-15C, and prostaglandin
I.sub.2.
18. The method of claim 16, wherein the beta-adrenergic receptor
agonist is selected from the group consisting of epinephrine and
isoproterenol.
19. The method of claim 13, further comprising co-administration
with insulin.
20. The method of claim 13, wherein blood flow of the subject is
increased.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 11/695,102, filed on Apr. 2, 2007, which claims priority
to U.S. provisional patent application No. 60/788,584, filed on
Apr. 1, 2006, all of which are herein incorporated by reference in
their entireties.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention is directed generally to compositions and
methods for controlling vascular caliber in extremities to improve
blood flow at extremities and control pulmonary blood pressure.
More specifically, the invention is directed to the control of ATP
release by red blood cells, via manipulating phosphodiesterase 3 in
red blood cells using inhibitors of phosphodiesterase 3.
[0004] 2. Description of the Related Art
[0005] It is known in the art that shear stress in arterioles
stimulates the production of endothelium relaxation factors that
act on the smooth muscle cells of arterioles, causing the vessels
to relax and permit increased blood flow. An example of an
endothelium relaxation factor is nitric oxide. Work performed by
the inventors and collaborators have suggested that red blood
cells, while under conditions of low oxygen tension or cell
deformation (e.g., as RBCS squeeze through smaller vessels),
release ATP into the blood vessel lumen. The ATP released from red
blood cells, in turn leads to NO synthesis and/or release of
endothelium derived relaxation factors by the endothelial cells,
which enables vasorelaxation.
[0006] It has also been shown by the inventors that patients with
certain diseases produce red blood cells which release none to
subnormal levels of ATP in response to low oxygen tension or under
mechanical deformation. Those diseases include (but are certainly
not envisioned to be limited to) cystic fibrosis, hyperinsulinemia,
prediabetes, metabolic syndrome, type 2 diabetes, and primary
pulmonary hypertension. The inventors envision that RBCS are an
important system for the regulation of blood flow into areas and
conditions of low oxygen tension, and subject to mechanical
deformities such as in capillary beds and at extremities. The
invention discloses (a) the mechanism by which RBCS can release
ATP, (b) compositions to enhance the production of ATP by RBCS, and
(c) methods of treating diseases such as pulmonary hypertension and
diabetic blood flow problems.
[0007] The erythrocyte, by virtue of the hemoglobin that it
contains, has long been recognized as a vehicle for oxygen
(O.sub.2) transport. In addition to this well established role for
the erythrocyte in the circulation, it has been shown that this
cell can also participate in the regulation of vascular resistance
via the release of ATP [2-8]. The erythrocyte releases ATP when
exposed to reduced O.sub.2 tension or mechanical deformation, as
well as in response endogenous mediators [1, 2, 4, 7, 9, 11, 13].
This erythrocyte-derived ATP has been shown to be a stimulus for NO
synthesis [3, 7, 8]. The ability of the erythrocyte to release ATP
in response to physiological stimuli enables this cell to control
its own distribution within the microcirculation and, thereby, to
regulate O.sub.2 delivery [2-4, 6]. Indeed, it has been proposed
that the erythrocyte, via its ability to release ATP in response to
reduced O.sub.2 tension, produces local vasodilation in areas of
skeletal muscle with increased O.sub.2 demand resulting in the
matching of O.sub.2 delivery with metabolic need [2, 5, 6, 30].
[0008] Recently, the inventors have defined a signal transduction
pathway that relates physiological and pharmacological stimuli to
ATP release from erythrocytes [12]. This pathway includes the
heterotrimeric G proteins Gi and Gs, adenylyl cyclase (AC), cyclic
adenosine monophosphate (cAMP), protein kinase A (PKA), and the
cystic fibrosis transmembrane conductance regulator (CFTR) (FIG.
10) [9-13]. It is important to note that, in this pathway,
activation of either Gs or Gi results in the stimulation of AC
activity and cAMP synthesis [9, 10]. The finding that activation of
Gi is capable of stimulating some AC subtypes is not unique to the
erythrocyte [31, 32]. It has been reported that, in multiple cell
types, AC type II is activated by the .beta.y subunit of Gi [10,
12, 14, 31, 32]. We have shown that both Gi and AC type II are
components of human erythrocyte membranes [10, 14].
[0009] As depicted in FIG. 10, increases in cAMP are required for
ATP release from erythrocytes [11]. The level of cAMP in a cell is
the product of its synthesis by AC and its degradation by
phosphodiesterases (PDEs) [33]. In addition to AC II, it is known
in the art that human (any and all) erythrocytes possess PDE
activity, however, neither the PDE subtypes present nor their
regulation have yet to be fully characterized [12, 14, 34-38].
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[0091] Sprague et al., Am. J. Physiol. Cell Physiol. 281: pp.
C1158-1164; 2001.
[0092] Lobato et al., Br. J. Anaesth 95: pp. 317-322; 2005.
[0093] Sprague et al., "Red Blood Cell-Derived ATP Is a Determinant
of Nitric Oxide Synthesis in the Pulmonary Circulation," Ch. 9 in
Interactions of Blood and the Pulmonary Circulation , Weir, Reeve
and Reeves (ed), Futura Publishing Co., Inc., Armonk, N.Y.;
2002.
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2002.
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pp. H693-700; 2003.
[0096] Olearczyk et al., Am. J. Physiol. Heart Circ. Physiol. 286:
pp. H940-945; 2004.
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1079-1084; 2004.
[0098] Olearczyk et al., Am. J. Physiol. Heart Circ. Physiol. 287:
pp. H748-754; 2004.
SUMMARY OF THE INVENTION
[0099] Having known that RBCS release ATP, and that pathway
involves the G-proteins Gi and Gs, adenylyl cyclase, protein kinase
A (PKA), and the cystic fibrosis transmembrane receptor (CFTR)
(FIG. 1), the inventors made the surprising discovery that RBCS
contain phosphodiesterase 3 (PDE3) and that PDE3 regulation can
potentiate ATP release by RBCS (FIGS. 2 and 3). Thus, in one
embodiment, the invention is directed to a method of enhancing the
production of ATP by RBCS, by contacting RBCS with an inhibitor of
PDE (e.g., IBMX), preferably a selective inhibitor of PDE3 (e.g.,
milrinone or cilostazol), more preferably the selective PDE3
inhibitor cilostazol. The RBCS can be ex vivo or in vivo, wherein
in vivo includes within a human patient.
[0100] Given that it is known that patients suffering from
pulmonary hypertension, cystic fibrosis, and having newly disclosed
in this application that patients suffering from hyperinsulinemia,
prediabetes, metabolic syndrome and/or type 2 diabetes have RBCS
that fail to release optimal amounts of ATP, the inventors envision
that PDE inhibitors can be indicated for patients suffering from
those diseases, to reduce vascular resistance and the increase
blood flow to extremities. Thus, in another embodiment, the
invention is directed to treating patients suffering from vascular
diseases associated with cystic fibrosis, pulmonary hypertension,
hyperinsulinemia, prediabetes, metabolic syndrome, diabetes, or the
like by administering a therapeutic amount of PDE inhibitor to the
patient. The USP and Physicians Desk Reference list PDE inhibitors
that are presently approved for use in humans (pre-existing art).
Those PDE inhibitors thus are preferred. However, improved PDE
inhibitors, especially improved PDE3 inhibitors can be used in the
practice of this invention. The preferred PDE3 inhibitor is
cilostazol, which is currently indicated for peripheral vascular
disease.
[0101] The inventors have also made the surprising discovery that
RBCS from patients suffering from diabetes, namely type 2 diabetes,
release suboptimal amounts of ATP in response to low oxygen tension
and mechanical deformation (FIG. 4). The G-protein, Gi, which is
critical in the formation and release of ATP by RBCS, was
discovered to be reduced in diabetic RBCS (FIG. 5; black bars
represent diabetic RBCS, white bars represent normal RBCS; western
blot strip on left represent normal RBCS, those on the right are
diabetic). It is known in the art that mastoparan 7 (MAS7)
stimulates Gi, which in the case of RBCS, in turn stimulates
adenylyl cyclase to produce cAMP and eventually increased ATP
release. When diabetic cells were treated with MAS7 and ATP release
was measured, the inventors observed that ATP release decreased
with an increase in hemoglobin glycation (hemoglobin Alc; FIG. 6),
thus demonstrating the correlation between blood sugar and
decreased ATP release. Knowing that (a) PDE3 is present in RBCS,
(b) PDEs catalyze the hydrolysis of cAMP to form AMP, and thus
reduce the pool of cAMP available for stimulation of ATP release
via activation of PKA, and (c) inhibitors of PDE can increase the
pool of cAMP available for stimulation of ATP release, the
inventors treated diabetic RBCS with the PDE3-specific inhibitor
cilostazol (CILO, FIG. 7) and looked for a change in MAS7-induced
ATP release. FIG. 7 summarizes those observations and clearly
demonstrates the CILO-treated diabetic RBCS can release amounts of
ATP not significantly different from the levels released by RBCS of
healthy individuals upon stimulation via the Gi pathway. Thus, as
described above, the inventors envision that PDE3 inhibitors
(preferably cilostazol) can be administered to patients suffering
from diabetes (particularly type 2 diabetes, but not excluding the
potential for type 1 diabetes) to increase the release of ATP by
RBCS in low oxygen tension conditions and under mechanical
deformation, to increase blood flow to extremities.
[0102] It is known in the art that RBCS have insulin receptors,
which has been exploited as a tool for studying insulin binding.
However, the role of insulin in RBC activity has not been
elucidated. The inventors have made the additional surprising
discovery that insulin affects the Gi-mediated release of ATP by
RBCS (summarized in FIG. 10). FIGS. 8 and 9 depict that RBCS
incubated in the presence of insulin show a decline in Gi-induced
cAMP production and ATP release (respectively). Thus, in another
embodiment, the invention is directed to treatment protocols for
prediabetes and type 2 diabetes, comprising administering to a
patient a therapeutically effective amount of a PDE3-specific
inhibitor (preferably cilostazol), thereby improving blood flow to
extremities and decreasing complications associated with peripheral
vascular disease.
[0103] Having made the discoveries that RBCS contain active PDE3,
which impinges on the ATP release pathway of RBCS, which affects
vasorelaxation, the inventors envision that this can be used as a
screening tool for drugs that affect blood flow to extremities,
vasorelaxation and contraction, peripheral hypertension and
pulmonary hypertension. Thus, in yet another embodiment, the
invention is directed to a drug screening platform and methods for
screening for those types of drugs, comprising contacting RBCS
(normal, cystic fibrosis, diabetic, primary pulmonary hypertensive,
human, rabbit, dog and the like) with a prospective drug candidate,
stimulating the RBC via the Gi pathway (e.g., low oxygen,
mechanical deformation, MAS7, and/or the like), and measuring ATP
release.
[0104] The inventors have also made the surprising discovery that a
combination of prostaglandins (or prostaglandin analogues; a subset
of which are prostacyclins and prostacyclin analogues),
beta-adrenergic receptor agonists (e.g., epinephrine,
isoproteranol) and PDE inhibitors provide a combinatorial or
synergistic effect on ATP release by erythrocytes. Example
prostaglandins and analogues include, but are not limited to
iloprost, UT-15C, prostaglandin I 2 (PGI 2). Examples of PDE
inhibitors include pentoxifylline (PTOX), IBMX, milrinone, and
cilostazol. FIGS. 17 and 18 show the effect of prostaglandin
analogues on cAMP production in rabbit and human erythrocytes,
respectively. FIGS. 12-16 depict the effects of prostaglandin
analogues in combination with PDE inhibitors on ATP production by
RBCS. Thus, in another embodiment, the invention is directed to
compositions, which are useful for increasing ATP production or
release by erythrocytes, comprising prostaglandins or analogues
thereof and PDE inhibitors. In an alternative embodiment, the
invention is also directed to methods of treating diseases
associated with increased vascular resistance, including but not
limited to cystic fibrosis, pulmonary hypertension, and
hyperinsulinemia, prediabetes, metabolic syndrome, type 2 diabetes.
In both of these embodiments, a preferred prostaglandin analogue is
iloprost and a preferred PDE inhibitor is a PDE3 inhibitor,
cilostazol.
BRIEF DESCRIPTION OF THE DRAWINGS
[0105] The drawings and concomitant descriptions are incorporated
into this disclosure.
[0106] FIG. 1 depicts a cartoon red blood cell and the cell
signaling pathway involved in ATP release from the red blood
cell.
[0107] FIG. 2 depicts a cartoon red blood cell and the cell
signaling pathway involved in ATP release from the red blood cell,
with the novel addition of phosphodiesterase 3B.
[0108] FIG. 3 depicts a portion of a western blot showing PDE3B
present in the membranes of human red blood cells.
[0109] FIG. 4 depicts the release of ATP by RBCS under reduced
oxygen tension, showing the reduced ATP release by RBCS from humans
suffering from type 2 diabetes.
[0110] FIG. 5 depicts western blots and quantified data for the
expression of various depicted isoforms of G-protein. RBCS from
patients suffering from type 2 diabetes show reduced levels of
Gi.sub.2.
[0111] FIG. 6 depicts the amount of RBC released ATP via Mas7
stimulation versus glycosylated hemoglobin, which represents
diabetic RBCS. Reduced levels of ATP are released from diabetic
RBCS.
[0112] FIG. 7 depicts cAMP levels in type 2 diabetic RBCS
stimulated with Mas7. The addition of the PDE3 inhibitor cilostazol
enables the diabetic RBCS to release normal levels of ATP under
Mas7 stimulation.
[0113] FIG. 8 depicts cAMP produced by RBCS treated with insulin.
Insulin reduces the amount of cAMP produced by RBCS under Mas7 (Gi)
stimulation.
[0114] FIG. 9 depicts ATP released by RBCS treated with insulin.
Insulin reduces the amount of ATP released by RBCS under Mas7 (Gi)
stimulation.
[0115] FIG. 10 depicts a cartoon red blood cell and the cell
signaling pathway involved in ATP release from the red blood cell,
with the novel addition of phosphodiesterase 3B and the insulin
receptor.
[0116] FIG. 11 depicts the effects of cilostazol on iloprost
stimulation of cAMP accumulation in human RBCS.
[0117] FIG. 12 depicts the effects of IBMX on iloprost stimulation
of cAMP accumulation in rabbit RBCS.
[0118] FIG. 13 depicts the effects of pentoxifylline on iloprost
stimulation of cAMP accumulation in rabbit RBCS.
[0119] FIG. 14 depicts the effects of milrinone on iloprost
stimulation of cAMP accumulation in rabbit RBCS.
[0120] FIG. 15 depicts the effects of cilostazol on iloprost
stimulation of cAMP accumulation in rabbit RBCS.
[0121] FIG. 16 depicts the effects of rolipram on iloprost
stimulation of cAMP accumulation in rabbit RBCS.
[0122] FIG. 17 depicts the effects of the PGI.sub.2 analogues
iloprost and UT-15C on cAMP accumulation in rabbit RBCS.
[0123] FIG. 18 depicts the effects of the PGI 2 analogue iloprost
on cAMP accumulation in human RBCS.
[0124] FIG. 19 depicts the effect of cilostazol on Mas7-induced
cAMP increases in RBCS derived from humans having type 2
diabetes.
[0125] FIG. 20 depicts the effect of cilostazol on Mas7-induced ATP
release by RBCS derived from humans having type 2 diabetes.
DETAILED DESCRIPTION OF THE INVENTION
[0126] The oxygen (O.sub.2) required to meet the metabolic needs of
all tissues is delivered by the erythrocyte, a small, flexible cell
containing hemoglobin which, in mammals, is devoid of a nucleus and
mitochondria. Recently, it has been demonstrated that this cell is
significantly more than an O.sub.2 transporter, but rather is a
complex cell that controls its own distribution within the
microcirculation via its ability to release adenosine triphosphate
(ATP) in response to reduced O.sub.2 tension [1-5].
Erythrocyte-derived ATP stimulates the synthesis of
endothelium-derived vasodilators resulting in increases in blood
flow, and, thereby, erythrocyte (O.sub.2) supply rate permitting
this cell to deliver oxygen in amounts required to precisely meet
local metabolic need [5-8]. A signal-transduction pathway that
relates ATP release to physiological and pharmacological stimuli
has been defined and includes the heterotrimeric G proteins Gs and
Gi, adenylyl cyclase (AC), protein kinase A (PKA), and the cystic
fibrosis transmembrane conductance regulator (CFTR) (FIG. 10)
[9-13]. Importantly, increases in cyclic adenosine monophosphate
(cAMP) are required for ATP release from erythrocytes suggesting
that regulation of the concentration of cAMP could be a critical
control point in this pathway [11, 12].
[0127] In the vasculature of skeletal muscle, failure of the
erythrocyte to release ATP in response to reduced O.sub.2 tension
could be expected to lead to impaired matching of O.sub.2 delivery
with metabolic need and, thereby, contribute to vascular disease.
In support of this hypothesis, the inventors have discovered that
ATP release from erythrocytes of humans with type 2 diabetes is
impaired [14], suggesting that this defect in erythrocyte
physiology could contribute to the associated vascular disease. It
has been reported that vascular complications of type 2 diabetes
are present before diagnosis [15], i.e., during the prediabetic
period when normal blood glucose levels are maintained at the
expense of marked hyperinsulinemia [16]. It has been previously
demonstrated that in prediabetes, increased plasma insulin levels
correlate with the degree of microvascular dysfunction, supporting
a connection between increased insulin levels and the development
of vascular disease [17, 18]. In several tissues, insulin has been
shown to activate a signaling pathway involving PI3 kinase (PI3K)
that activates phosphodiesterase 3 (PDE3) to hydrolyze cAMP
[19-23]. In the erythrocyte, increased hydrolysis of cAMP would
impair ATP release in response to physiological stimuli such as
exposure to reduced O.sub.2 tension as well as in response to
pharmacological stimuli (FIG. 1). Human erythrocytes possess
insulin receptors and phosphoinositol 3 kinase [24-27], however,
neither insulin signaling pathways nor PDE activity in the
erythrocyte have been fully characterized.
[0128] The inventors herein describe a discovery in which levels of
insulin of the magnitude found in humans with prediabetes can
activate PDE3 in human erythrocytes resulting in reduced cAMP
accumulation and decreased ATP release in response to physiological
and pharmacological stimuli [28, 29]. Moreover, while not wishing
to be bound by theory, the inventors reasonably postulate that
these effects of insulin are the result of receptor-mediated
activation of insulin signaling pathways in the erythrocyte and
that the adverse effects of insulin can be attenuated by PDE3
inhibition providing a new rational for the use of PDE3 inhibitors
in diabetic vascular disease. The inventors have demonstrated that
(1) insulin decreases cAMP accumulation in human erythrocytes and,
consequently, attenuates ATP release in response to activation of
the heterotrimeric G protein Gi as well as the physiological
stimulus of exposure to reduced O.sub.2 tension, (2)
insulin-induced inhibition of erythrocyte cAMP accumulation and ATP
release is mediated via activation of the insulin receptor
resulting in the stimulation of a signaling pathway involving
PI3K/PKB and, ultimately, activation of PDE3, and (3) inhibition of
the activity of PDE3 attenuates insulin-induced decreases in cAMP
accumulation and ATP release from human erythrocytes in response to
physiological and pharmacological stimuli.
[0129] Phosphodiesterase isoenzymes (PDEs) are present in every
mammalian cell and at least 11 families of PDEs, some with multiple
isozymes, have been described [33]. Different PDE families
hydrolyze cAMP, cyclic guanosine monophosphate (cGMP) or both
cyclic nucleotides [33]. One of these families, PDE3, comprising
PDE3A and PDE3B, has been found to be important in regulating such
diverse effects of cAMP as lipolysis in adipocytes, glycogen
metabolism in liver, apoptosis of cardiomyocytes, aggregation of
platelets, and insulin release by [3 cells [23, 33, 39-42].
Interestingly, insulin, acting through the insulin receptor and
associated signaling, has been shown to activate PDE3 in adipocytes
enabling insulin to antagonize the effects of cAMP in this cell.
The signaling pathway for insulin-induced PDE3 activation requires
the tyrosine kinase activity of the insulin receptor,
phosphoinositide 3-kinase (PI3K), phosphoinositide-dependent
kinases (PDKs), and protein kinase B (PKB) which ultimately
phosphorylates serine residues on PDE3B resulting in its activation
[20-22, 43-45].
[0130] Human erythrocytes possess insulin receptors and have been
used to characterize insulin receptor structure and binding
characteristics [25, 26, 46, 47]. Although tyrosine kinase activity
is present in erythrocytes [48], insulin signaling in this cell has
not been studied. Erythrocytes do not require insulin for glucose
uptake. However, insulin has been reported to increase calcium flux
[49], possibly reflecting insulin signaling in that cell. No
studies have addressed the effect of insulin on erythrocyte cAMP
levels or ATP release. If insulin signaling does occur in the
erythrocyte, one consequence could be the activation of PDE3
leading to increased hydrolysis of intracellular cAMP and
inhibition of ATP release in response to physiological and
pharmacological stimuli. The inventors have discovered and disclose
herein that there is an insulin-mediated effect on ATP release.
[0131] In addition to insulin receptors, erythrocytes possess
receptors for insulin-like growth factor type 1 (IGF-1) to which
insulin binds with low affinity [50, 51]. Additionally, the IGF-1
receptor on [3 cells has been demonstrated to activate PDE3B [23,
52]. The binding of insulin to either receptor could result in
activation of PI3K and, consequently, PDE3B [50]. Importantly, in
human erythrocytes, insulin receptors are present in vastly greater
numbers than are IGF-1 receptors [24, 46, 51, 53] suggesting that
any receptor-mediated effect of insulin is mediated via the insulin
receptor.
[0132] In humans with prediabetes, high insulin levels maintain
normal blood glucose levels during the initial development of
insulin resistance [16]. Importantly, microvascular dysfunction in
these individuals correlates with plasma insulin levels [17, 18],
suggesting a potential pathophysiological consequence of sustained
increases in plasma insulin. One reasonably expected consequence of
the increased plasma insulin levels in prediabetes could be
increased activation of PDE3 within the erythrocyte resulting in
enhanced cAMP hydrolysis and, consequently, decreased ATP release
in response to exposure to reduced O.sub.2 tension. Thus,
hyperinsulinemia, via this mechanism, could contribute to the
failure to match O.sub.2 delivery with metabolic need in the
skeletal muscle microcirculation and contribute to the vascular
complications of prediabetes. In this disclosure, the inventors
determined that insulin, acting through receptor-mediated insulin
signaling, activates (in some way, as determined via PDE
inhibition) PDE3, leading to decreased cAMP levels and impaired ATP
release from erythrocytes in response to exposure to reduced
O.sub.2 tension as well as in response to pharmacological
activation of the heterotrimeric G proteins Gs and Gi.
[0133] If insulin-induced activation of PDE3 and subsequent
hydrolysis of cAMP is an important contributor to abnormal
erythrocyte physiology, it is reasonable to assume that
pharmacological agents that decrease PDE3 activity could hold
promise as therapeutic agents for the microvascular disease
associated with hyperinsulinemia, prediabetes, metabolic syndrome,
and type 2 diabetes. Currently, an agent that is a relatively
selective PDE3 inhibitor, cilostazol is approved for use in the
treatment of intermittent claudication [54], a condition in which
O.sub.2 supply to skeletal muscle is inadequate to meet metabolic
need. However, the mechanism by which cilostazol improves the
symptoms of claudication has not been defined. The inventors herein
establish and disclose that inhibitors of PDE3 activity increase
cAMP levels in erythrocytes and facilitate ATP release in response
pharmacological mediators as well as exposure of erythrocytes to
reduced O.sub.2 tension. This important and useful discovery
provides a heretofore novel mechanism of action of PDE3 inhibitors
used clinically in vascular disease and suggests a new indication
for these agents in the treatment of the microvascular disease of
diabetes. Thus, the invention is directed to compositions (PDE3
inhibitors, direct and indirect) and methods of treating
microvascular disease, by employing a PDE3 inhibitor in a
pharmaceutically acceptable excipient.
[0134] It is important to recognize that, in the study of the
mature erythrocyte, the use of many molecular tools is precluded.
Erythrocytes lack a nucleus and protein synthesis capability. Thus,
in yet another embodiment, the inventors have developed an
experimental approach (kits, tools and process) that utilizes
physiological stimuli as well as traditional pharmacological
approaches coupled with activity assays and Western analysis of
erythrocyte membrane fractions to investigate the role of PDE3 and
its regulation by insulin and pharmacological agents in ATP release
from erythrocytes. These methods are exemplified in the examples
that follow.
EXAMPLE 1
PDE3 in RBCS and the Effect of Insulin on ATP Release by RBCS
[0135] Erythrocytes possess the heterotrimeric G proteins Gi and Gs
as well as adenylyl cyclase. Importantly, activation of either of
these G proteins results in stimulation of adenylyl cyclase and the
synthesis of cAMP. Increases in cAMP are required for ATP release
from erythrocytes. ATP released from erythrocytes in the vascular
lumen is a stimulus for nitric oxide synthesis. Incubation of
erythrocytes with Mastoparan 7, a compound that activates Gi,
results in increases in cAMP and ATP release (Am. J. Physiol. Heart
Circ. Physiol. 287(2): H748-H754, 2004, which is herein
incorporated by reference.) The concentration of cAMP in cells is
dependent on the rate of its synthesis as well as its degradation
by phosphodiesterases (PDEs). Previous studies demonstrate that
PDE3 activity is increased by insulin and inhibited by cilostazol
in a number of cell types (Cell. Signal. 7(5): 445-455, 1995).
Erythrocytes have a well characterized insulin receptor; however,
it has not been determined if the mature erythrocyte possesses
PDE3. Here, we investigate the hypothesis that human erythrocytes
possess PDE3 and that insulin decreases intracellular cAMP, thereby
inhibiting ATP release from these cells.
Isolation of Human Erythrocytes
[0136] Human blood was obtained by venipuncture. Blood (35 ml) was
collected in a syringe containing heparin (500 units) and
centrifuged at 500.times.g for 10 min at 4.degree. C. The plasma,
buffy coat, and uppermost erythrocytes were removed by aspiration
and discarded. The remaining erythrocytes were washed three times
in buffer (in mM; 21.0 Tris-HCl, 4.7 KCl, 2.0 CaCl.sub.2 , 140.5
NaCl, 1.2 MgSO.sub.4 with 2.5% dextrose and 0.5% bovine serum
albumin, fraction V, final pH adjusted to 7.4). After the last
centrifugation, the hematocrit of the erythrocytes was determined.
The protocol for blood collection was approved by the Institutional
Review Committee of Saint Louis University.
Incubation of Erythrocytes with Pharmacological Agents
[0137] Erythrocytes (20% hct) were incubated with Mastoparan 7 (10
.mu.M) or its vehicle in the presence or absence of 1 .mu.M
insulin. ATP release from erythrocytes was measured at 5 minute
intervals using the luciferin/luciferase assay. In separate
studies, erythrocytes (50% hct) were incubated with vehicle, 10
.mu.M cilostazol, 100 .mu.M cilostazol, 1 .mu.M insulin, 3 .mu.M
insulin, and/or 10 .mu.M Mastoparan 7 for determination of cAMP
concentration. At timed intervals, 4 mL of ice cold ethanol
containing 1 mM HCl was added to 1 mL of RBC suspension. After
centrifugation at 14,000.times.g at 4.degree. C. for 10 min, the
supernatant was dried under vacuum. Samples were resuspended in
assay buffer and cAMP concentration was determined in duplicate by
EIA.
Western Blot Analysis
[0138] Erythrocyte membranes were prepared by lysis of erythrocytes
in buffer containing 5 mM Tris-HCl and 2 mM EDTA with pH adjusted
to 7.4. Erythrocyte membranes were then isolated by centrifugation
at 23,700.times.g at 4.degree. C. for 10 min. Protein concentration
in the pellet was determined by BCA assay. Membrane proteins were
solubilized in SDS sample buffer (8% SDS, 60% glycerol, 0.25 M Tris
HCl (pH 6.8), 0.004% bromophenol blue, and 400 mM dithiothreitol)
and boiled for 5 min before loading onto a 5% gel. After
electrophoresis, proteins were transferred onto a PVDF membrane in
buffer containing 25 mM Tris-base, 192 mM glycine, and 10%
methanol. The PVDF membranes were then blocked overnight with 5%
non-fat dry milk in phosphate-buffered saline containing 0.1%
Tween-20. Membranes were immunoblotted with an antibody directed
against amino acids 1-300 of human PDE3B. Membranes were then
incubated with donkey anti-rabbit IgG-horseradish peroxidase and
protein was visualized using enhanced chemiluminescence.
Data Analysis
[0139] Values are mean.+-.SEM. Statistical significance was
determined by ANOVA followed by Fisher's LSD or paired T-test, as
appropriate.
Summary
[0140] PDE3B has been identified, for the first time, in the
membrane of human erythrocytes. In the presence of cilostazol, a
phosphodiesterase 3 inhibitor, basal cAMP levels are increased in
human erythrocytes. Mastoparan 7, compound that activates Gi,
results in cAMP production and ATP release from human erythrocytes.
Incubation of erythrocytes with insulin results in decreases in
both Mas7-induced cAMP accumulation and ATP release.
Conclusion
[0141] These findings support previous studies demonstrating that
Mastoparan 7, by activating Gi in the human erythrocyte, stimulates
adenylyl cyclase resulting in increased cAMP production and ATP
release from these cells. Release of ATP has been suggested to be
an important factor in matching blood flow with metabolic need in
skeletal muscle. In the work presented here we show that, in human
erythrocytes, insulin reduces both cAMP production and ATP release
in response to Mastoparan 7. These findings are consistent with the
hypothesis that PDE3B, present in human erythrocytes, can be
activated by insulin. One interpretation of these results is that
increased plasma insulin could contribute to the vascular
complications of pre-diabetes and diabetes.
[0142] We postulate that this effect of insulin is modulated via
the following signal transduction pathway.
EXAMPLE 2
[0143] Insulin decreases cAMP accumulation in human erythrocytes
and, consequently, attenuates ATP release in response to exposure
to reduced O.sub.2 tension as well as pharmacological activation of
the heterotrimeric G proteins Gs and Gi. Incubation of erythrocytes
of humans with mastoparan 7 (MAS7), a direct activator of Gi,
results in cAMP accumulation and ATP release [10, 12]. In other
cells, insulin has been has been shown to decrease cAMP levels, a
requisite for erythrocyte ATP release, by activating
phosphodiesterase 3 (PDE3) [20, 23].
[0144] The inventors have shown that pre-incubation of human
erythrocytes with insulin (1 .mu.M) attenuated MAS7 (10
.mu.M)-induced ATP release in the absence of changes in total ATP
content (FIG. 9). Importantly, insulin pre-treatment also resulted
in inhibition of MAS7 (10 .mu.M)-induced cAMP accumulation (FIG.
8). These results provide support for the hypothesis that insulin,
at concentrations present in humans with pre-diabetes, inhibits
cAMP accumulation, a requisite for ATP release from erythrocytes,
possibly via stimulation of cAMP hydrolysis [28, 29].
[0145] Protocol A: Washed erythrocytes were incubated with insulin
(0.1 nM-1 .mu.M, Humalog.RTM.), or its vehicle (saline) for 30 min
[28, 29, 47, 60]. Erythrocytes were then incubated with either
iloprost (1 .mu.M), isoproterenol (10 .mu.M) (receptor-mediated
activators of Gs) or mastoparan 7 (MAS7, 10 .mu.M) (a direct
activator of Gi) or their vehicle (saline) [9, 10, 12, 57, 61].
After 15 min, cAMP accumulation and ATP release will be
determined.
[0146] For controls: Humalog.RTM. is a human insulin analog with
the reversal of the position of two amino acid residues at the C
terminal of the B chain. This modification results in an insulin
that displays a decreased tendency to dimerize and therefore
retains its activity under in vitro conditions. Still, Humalog.RTM.
is equipotent to human insulin on a molar basis (product insert,
Eli Lilly). Under all protocols, a limited number of identical
experiments are conducted using regular human insulin
(Humulin.RTM., Eli Lilly) in lieu of Humalog.RTM. to establish that
any effect of Humalog.RTM. is not the result of this structural
alteration. Finally, total intracellular ATP and free hemoglobin, a
measure of erythrocyte lysis, are determined (infra).
[0147] Protocol B: To determine the effects of insulin on
erythrocyte cAMP accumulation and ATP release induced by decreased
O.sub.2 tension, washed erythrocytes are incubated with insulin
(0.1 nM-1 .mu.M) or its vehicle (saline) for 30 min. Erythrocytes
are then placed in a tonometer (infra) and exposed to gas
containing 15% O.sub.2, 5% CO.sub.2; 0% O.sub.2, 5% CO.sub.2,
(normoxia, pO.sub.2.apprxeq.100 mm Hg). The pH, pCO.sub.2 and
pO.sub.2 as well as cAMP or ATP concentration were determined at 30
min after addition of the erythrocyte suspension to the tonometer.
The gas tension was then changed to 4.5% O.sub.2, 5% CO.sub.2; 0%
O.sub.2, 5% CO.sub.2, (hypoxia, pO.sub.2.apprxeq.20 mm Hg) and pH,
gas tensions, cAMP and ATP measurements are repeated at 5, 10, and
15 min. Finally, the gas tension is returned to the "normoxic" gas
composition (recovery) and the measurements are at 30 min.
[0148] Protocol C: To determine effects of direct activation of
adenylyl cyclase and active cAMP analogs on ATP release from
erythrocytes in the presence of insulin, washed erythrocytes are
incubated with insulin (0.1 nM-1 .mu.M) or its vehicle (saline).
After 30 min, the cells are incubated with either the non-selective
activator of adenylyl cyclase (AC) activity, forskolin (1 to 10
.mu.M) [11], or one of two active cAMP analogs that are resistant
to degradation, SpcAMP (10 to 100 .mu.M) [11] or 8-bromo-cAMP (10
to 100 .mu.M) [62]. ATP release was measured at 5, 10 and 15 min
after addition of forskolin or the active cAMP analogs. Controls:
To ensure the effects of forskolin or the cAMP analogs are not due
to effects of vehicle or time, identical experiments are conducted
using their vehicles, N,N-dimethylformamide (DMF) or saline,
respectively, in lieu of the active agent. Total intracellular ATP
and free hemoglobin, a measurement of erythrocyte lysis, is
determined.
[0149] Results: Pharmacological activation of Gs (iloprost or
isoproterenol), as well as pharmacological (MAS7) and physiological
(decreased O.sub.2 tension) activation of Gi, stimulate cAMP
synthesis in and ATP release from human erythrocytes. Pretreatment
of erythrocytes with insulin, at concentrations reported to be
present in the plasma of humans with prediabetes, inhibited both
cAMP accumulation and ATP release from human erythrocytes. These
effects of insulin reflect negative cooperative binding of insulin
with its receptor, i.e., nM concentrations of insulin will be more
effective. The findings that insulin inhibits both Gs- and
Gi-mediated ATP release but has no effect on ATP release in
response to active cAMP analogs that are resistant to hydrolysis by
PDEs provides strong support that insulin acts to decrease ATP
release via activation of PDE activity in the human erythrocyte and
not on other components of the signal transduction pathway (FIG. 1,
above).
EXAMPLE 3
[0150] Insulin-induced inhibition of erythrocyte cAMP accumulation
and ATP release is mediated via activation of the insulin receptor
resulting in the stimulation of a signaling pathway reasonably
expected to involve PI3K/PKB and, ultimately, activation of PDE3.
Although human erythrocytes have been shown to possess insulin
receptors [25, 26, 46, 47], a role in erythrocyte physiology has
not been defined. The preliminary results reported above (FIGS. 8,
9) suggest that insulin can inhibit both cAMP accumulation and ATP
release from erythrocytes. Importantly, the effect on cAMP
accumulation is greater at lower (nM) insulin concentrations (FIG.
8). The latter finding is consistent with the hypothesis that this
effect of insulin is receptor mediated. The binding of insulin to
its receptor displays negative cooperativity, such that the
dissociation rate of insulin accelerates with increased receptor
occupancy [55, 56]. The result is that lower concentrations of
insulin stimulate a greater response than do higher concentrations
[55, 56].
[0151] Insulin has been reported to produce receptor-mediated
activation of PDE3 in other cells resulting in decreases in cAMP
[20, 23]. Although PDE activity has been reported in the
erythrocyte [34-38], the PDEs involved and the pathways by which
they could be regulated have not been characterized. Using Western
analysis, we have determined for the first time that PDE3B, the PDE
activated by insulin in other cell types [19, 23], is a component
of the membranes of human erythrocytes (FIG. 3). Taken together,
these preliminary studies are consistent with the hypothesis that
inhibition of cAMP accumulation in human erythrocytes by insulin
could be mediated via activation of the insulin receptor and
subsequent activation of PDE3, a PDE that hydrolyzes cAMP.
[0152] In other tissues, insulin antagonizes cAMP action via
receptor-mediated activation of signaling pathways that results in
activation of PDE3 [20, 22, 23, 45, 52, 65, 66]. Such a signaling
pathway has been characterized in adipocytes and includes the
insulin receptor, IRS, PI3K, PDKs, PKB, and PP2A [20]. Insulin can
interact with two distinct receptors, the insulin receptor and the
IGF-1 receptor, both present in erythrocytes membranes and both
associated with activation of PDE3 in different cell types [19,
23-25, 46, 50-52].
[0153] Protocol A: To determine that tyrosine kinase activity is
required for insulin-induced decreases in cAMP accumulation and ATP
release in response to physiological and pharmacological stimuli,
washed erythrocytes are treated with insulin (0.1 nM-1 .mu.M) or
its vehicle (saline) for 30 min followed by the addition of 300
.mu.M hydroxy-2-naphthalenylmethylphosphonic acid (HNMPA-(AM)3), an
insulin receptor tyrosine kinase inhibitor [67, 68]. After 30 min,
the cells are incubated with either iloprost (1 .mu.M),
isoproterenol (10 .mu.M), MAS7 (10 .mu.M) or exposed to reduced
O.sub.2 tension (supra) and cAMP accumulation and ATP release is
determined. Control experiments: In separate studies the vehicles
for HNMPA-(AM)3, iloprost, isoproterenol and MAS7 (DMF or saline)
are used in lieu of the active agent.
[0154] Protocol B: To determine the effects of blockade of the
IGF-1 receptor on insulin-induced inhibition of cAMP accumulation
and ATP release in response to physiological and pharmacological
stimuli, washed erythrocytes are treated with a blocking antibody
specific for the IGF-1 receptor, .alpha.IR3 (1 .mu.g of IgG per 100
.mu.l of erythrocyte suspension for 3 hours) [51], in the presence
or absence of insulin (0.1 nM-1 .mu.M). After 30 min, the cells are
incubated with either iloprost (1 .mu.M), isoproterenol (10 .mu.M),
MAS7 (10 .mu.M) or exposed to reduced O.sub.2 tension (as described
under EXAMPLE 2) and cAMP accumulation and ATP release is
determined. Control experiments: To ensure the effects of
.alpha.IR3 are not due to nonspecific effects of the antibody,
identical experiments are conducted using pre-immune mouse serum in
lieu of .alpha.IR3. In addition, experiments with the various
vehicles are performed in lieu of the active agents.
[0155] Protocol C: To determine the effects of IGF-1 on erythrocyte
cAMP accumulation and ATP release in response to physiological and
pharmacological stimuli, washed erythrocytes are treated with
either IGF-1 (10 .mu.M-100 nM [51]) or its vehicle saline) for 30
min. The cells are then incubated with either iloprost (1 .mu.M),
isoproterenol (10 .mu.M), MAS7 (10 .mu.M) or exposed to reduced
O.sub.2 tension (as described under EXAMPLE 2) and cAMP
accumulation and ATP release are determined. In separate studies,
erythrocytes are pretreated with .alpha.IR3, as described in
Protocol A to inhibit any effects of IGF-1 resulting from binding
to the IGF-1 receptor. Control experiments: In separate studies the
vehicles for IGF-1, iloprost, isoproterenol and MAS7 (DMF or
saline) are used in lieu of the active agent.
[0156] Protocol D: To establish that the effects of insulin-induced
decreases in cAMP accumulation and ATP release are the result of
increased activity of the phosphodiesterase, PDE3, erythrocyte
membranes were incubated with insulin (0.1 nM-1 .mu.M) or its
vehicle (saline) in the absence or presence of the PDE3 selective
inhibitors cilostazol (10, 30, or 100 .mu.M) or milrinone (10 or 30
.mu.M) [39, 54, 69]. cAMP substrate were added to the isolated
membranes and amounts of hydrolyzed AMP (PDE activity) were
determined as described (infra). The ability of PDE3 inhibitors to
interfere with insulin-induced increases in PDE activity was
interpreted to mean that insulin activates PDE3 activity in this
system. Control experiments: Identical experiments were performed
with the vehicle for cilostazol and milrinone, DMF.
[0157] Protocol E: To determine that PI3K is required for
insulin-induced inhibition of cAMP accumulation and ATP release in
response to physiological and pharmacological stimuli, washed
erythrocytes are treated with insulin (0.1 nM-1 .mu.M) or its
vehicle (saline) for 30 min. Cells are then treated with either of
two PI3K inhibitors, LY294002 (1 .mu.M) or wortmannin (100 nM) [22,
27]. After 30 min, the cells are incubated with either iloprost (1
.mu.M), isoproterenol (10 .mu.M), MAS7 (10 .mu.M) or exposed to
reduced O.sub.2 tension (as described supra) and cAMP accumulation
and ATP release are determined. Control experiments: In separate
studies the vehicle for LY29002 and wortmannin (DMF) are used in
lieu of the active compounds.
[0158] Protocol F: To determine that PKB is required for
insulin-induced inhibition of cAMP accumulation and ATP release in
response to physiological and pharmacological stimuli, washed
erythrocytes are treated with insulin (0.1 nM-1 .mu.M) or its
vehicle (saline) for 30 min in addition to a PKB inhibitor
(Akti-1/2, 1 .mu.M-30 .mu.M) [70]. The cells are then incubated
with either iloprost (1 .mu.M), isoproterenol (10 .mu.M), MAS7 (10
.mu.M) or exposed to reduced O.sub.2 tension (as described supra)
and cAMP accumulation and ATP release are determined. Control
experiments: In separate studies the vehicle (DMF) for Akti-1/2 is
used in lieu of the active compound.
[0159] Results: This example is expected to demonstrate that, in
the erythrocyte, insulin-induced inhibition of cAMP accumulation
and ATP release is mediated via activation of the insulin receptor.
In support of this hypothesis, it is demonstrated that inhibition
of tyrosine kinase activity prevents the actions of insulin since
this activity is required for the insulin receptor to initiate
downstream signaling. It is reasonably expected that blockade of
the IGF-1 receptor does not prevent the ability of insulin to
inhibit cAMP accumulation and ATP release. The use of the PDE
activity helped to establish that the ability of insulin to
decrease cAMP accumulation is related to the stimulation of PDE3.
Finally, inhibition of either PI3K or PKB is expected to prevent
activation of PDE3 by insulin, suggesting that both are components
of an insulin signaling pathway for activation of PDE3 in
erythrocytes, as has been reported in other cell types. Additional
support of a role for PKB was obtained by the demonstration that
inhibition of PKB prevents the activation of PDE3 and, thereby
potentiates cAMP accumulation and ATP release in response to
pharmacological stimuli. Taken together, these experiments are
expected to demonstrate that insulin bound to the erythrocyte
insulin receptor stimulates the activation of PDE3 in the
erythrocyte via a signaling pathway that involves PI3K and PKB.
EXAMPLE 4
[0160] Inhibition of the activity of PDE3 attenuates
insulin-induced decreases in cAMP accumulation and ATP release from
human erythrocytes in response to physiological and pharmacological
stimuli. The inventors had demonstrated that PDE3B is present in
erythrocyte membranes (supra). In this example, a role for this PDE
in the regulation of cAMP levels in human erythrocytes is
demonstrated.
[0161] To begin to address this issue, studies were performed in
which erythrocytes were pre-incubated with cilostazol, a well
characterized inhibitor of PDE3 activity [54], and then exposed to
iloprost, a prostacyclin analog that produces receptor-mediated
activation of Gs and increases in cAMP in erythrocytes [9, 12, 57].
As depicted in FIG. 11, cilostazol pre-treatment resulted in
concentration-dependent increases in iloprost-induced cAMP
accumulation. In addition to effects on PDE3, cilostazol has been
reported to inhibit PDE4 activity [54], but at concentrations
ten-fold greater than those used here. Importantly, the inventors
have established that the selective inhibitor of PDE4 activity,
rolipram (30 .mu.M, n=6) [33], has no effect on iloprost-induced
cAMP accumulation in human erythrocytes suggesting that the effects
of cilostazol are the result of selective inhibition of PDE3
activity.
[0162] Protocol A: To confirm that the protein identified by
Western analysis is PDE3B and that it is present in the erythrocyte
membrane, purified erythrocytes membranes were subjected to Western
analysis as described in General Methods (infra) using multiple
antibodies directed against different epitopes of PDE3B. Control
experiments: To establish that membrane preparations are devoid of
platelets which contain an isoform of PDE3, the membrane
preparations were probed for CD41, a protein found in platelets,
but not erythrocytes [40, 72]. In addition, lack of leukocyte
contamination was determined by microscopic examination of
erythrocyte preparations.
[0163] Protocol B: To demonstrate that selective PDE3 inhibitors
and non-hydrolysable cGMP analogs increase erythrocyte cAMP
accumulation and ATP release in response to physiological and
pharmacological stimuli, washed erythrocytes were incubated with
either of the active cGMP analogs, 8-BromocGMP or SpcGMP (50 or 100
.mu.M) or the selective PDE3 inhibitors cilostazol (10, 30, or 100
.mu.M) or milrinone (10 or 30 .mu.M) [39, 54, 69]. After 30 min,
the cells were incubated with either iloprost (1 .mu.M),
isoproterenol (10 .mu.M), MAS7 (10 .mu.M) or exposed to reduce
O.sub.2 tension and cAMP accumulation and ATP release was
determined. Control experiments: Identical experiments were
performed with the vehicles for the various inhibitors. In
addition, since PDE4 and PDE5 can be inhibited by high
concentrations of milrinone and cilostazol, separate studies were
performed in which erythrocytes were incubated with rolipram (30
.mu.M), a highly selective PDE4 inhibitor (see FIG. 16) [33, 39],
or sildenafil (10 nM; not yet done at time of filing), a PDE 5
inhibitor [33, 54] prior to physiological and pharmacological
stimulation. The concentrations chosen for rolipram and sildenafil
are those reported to be specific for the PDEs indicated.
[0164] Protocol C: To demonstrate that PDE3 inhibitors and
non-hydrolysable cGMP analogs oppose insulin-induced inhibition of
erythrocyte cAMP accumulation and ATP release in response to
physiological and pharmacological stimuli, washed erythrocytes were
incubated with either 8-Bromo-cGMP, SpcGMP, cilostazol or milrinone
[33, 54] along with the insulin at the concentrations determined to
be most effective for inhibition of cAMP accumulation and ATP
release. After 30 min, the cells were incubated with either
iloprost (1 .mu.M), isoproterenol (10 .mu.M), MAS7 (10 .mu.M) or
exposed to reduced O.sub.2 tension and cAMP accumulation and ATP
release were determined.
[0165] Results: The presence of PDE3 in the erythrocyte membrane
was demonstrated by Western analysis (FIG. 3) and suggests a novel
role for this PDE in erythrocyte physiology. PDE3 was shown herein
to play an important role in the regulation of cAMP levels leading
to alterations in ATP release. It was shown herein that insulin
inhibits ATP release via its ability to stimulate PDE3 activity and
limit cAMP accumulation. It was also shown herein that inhibition
of PDE3 activity increased cAMP accumulation and ATP release in
response to physiological and pharmacological stimuli. The
inventors conclude that inhibitors of PDE3 prevent insulin-induced
reductions in cAMP accumulation and ATP release in response to
physiological and pharmacological stimuli. Thus, it is reasonable
to expect that the inhibition of PDE3 activity presents a novel
approach to the prevention and treatment of the vascular
complications associated with prediabetes.
EXAMPLE 5
General Methods
[0166] Generation of Washed Erythrocytes: Human blood was obtained
by venipuncture in a syringe containing heparin (500 units) and was
centrifuged at 500.times.g for 10 minutes at 4.degree. C. The
plasma, buffy coat, and uppermost erythrocytes were removed by
aspiration and discarded. The remaining erythrocytes were washed
three times in buffer containing 21.0 mM Tris-HCl, 4.7 KCl, 2.0 mM
CaCl.sub.2, 140.5 mM NaCl, 1.2 mM MgSO.sub.4, 0.1% dextrose, and
0.5% bovine albumin fraction V, final pH 7.4. The hematocrit of the
washed erythrocytes was determined.
[0167] Preparation of Erythrocyte Membranes: Washed erythrocytes
were diluted 1:100 with ice cold buffer containing 5 mM Tris-HCl, 2
mM EDTA, pH 7.4, and stirred at 4.degree. C. for 15 minutes. The
lysate was centrifuged at 30,000.times.g for 15 minutes at
4.degree. C. The supernatant was removed and discarded. The pellet
containing the erythrocyte membranes was washed two times with
ice-cold buffer and centrifuged and the membranes were re-suspended
in ice cold buffer and frozen at -80.degree. C. Membrane protein
concentrations were determined using the BCA Protein Assay.
[0168] Preparation of Platelets for Western Analysis: Whole blood
was centrifuged at 400.times.g for 10 minutes at 4.degree. C. The
supernatant was collected and 0.5 ml heparin and 1 mg/ml EDTA were
added and re-centrifuged for 40 minutes at 200.times.g at 4.degree.
C. The platelet-rich-plasma was collected and centrifuged at
1,400.times.g for 20 minutes at 4.degree. C. The supernatant was
discarded and 200 ml of Western lysis buffer (25 mM HEPES, 300 mM
NaCl, 10 mM EDTA, 1.5 mM MgCl.sub.2, 6H.sub.2O, 20 mM
[3-glycerophosphate, 0.1 mM sodium vanadate, 1% Triton X-100) was
added to pellet followed by sonication (10 s) and, after 15 minutes
on ice, centrifugation at 14,000.times.g for 20 minutes at
4.degree. C. Platelet protein concentrations were determined using
the BCA Protein Assay.
[0169] Western Analysis: Erythrocyte membranes or platelets were
solubilized in SDS buffer (8% SDS, 60% glycerol, 0.25 M Tris HCl
(pH 6.8), 0.004% bromophenol blue, and 400 mM dithiothreitol),
boiled, and loaded onto a pre-cast 7.5% gel and subjected to
electrophoresis at 150 volts for 90 min and transferred to a
polyvinylidene difluoride (PVDF) membrane (100 volts for 60 min) in
buffer containing 25 mM Tris-base, 192 mM glycine, and 10%
methanol. Membranes were blocked overnight with 5% non-fat dry milk
in PBS containing 0.1% Tween-20, immunoblotted with a primary
antibody directed against protein of interest followed by
incubation with an appropriate secondary antibody in 1% non-fat dry
milk and visualized using enhanced chemiluminescence.
[0170] Measurement of ATP: ATP was measured using the
luciferin/luciferase assay. A 200 .mu.L sample of an erythrocyte
suspension was injected into a cuvette containing 100 .mu.L of 10
mg/ml crude firefly tail extract and 100 .mu.L of a 0.5 mg/ml
solution of D-luciferin. The light emitted from the reaction of ATP
with the crude firefly tail extract was measured using a
luminometer designed to detect a wavelength of 565 nm. The peak
light emitted was compared to an ATP standard curve generated on
the day of the experiment.
[0171] Measurement of Total Intracellular ATP: A known number of
erythrocytes was lysed in distilled water at room temperature. ATP
in the lysate, diluted 8,000 fold, was measured using ATP assay and
the values normalized to ATP concentration per erythrocyte.
[0172] Measurement of Hemoglobin: Erythrocyte suspensions used to
measure ATP were centrifuged at 500.times.g for 10 minutes at
4.degree. C. The amount of hemoglobin present in the supernatant
was determined by measurement of absorbance at 405 nm
(oxyhemoglobin).
[0173] Measurement of cAMP: 1 ml of erythrocyte suspension was
added to 4 ml of ice cold ethanol containing 1 mM HCl and the
mixture was centrifuged at 21,000.times.g for 10 minutes at
4.degree. C. The supernatant was removed and stored overnight at
-20.degree. C. to precipitate remaining proteins. Samples were
centrifuged a second time at 3,700.times.g for 10 minutes at
4.degree. C. The supernatant was removed and dried under vacuum
centrifugation. Concentrations of cAMP were determined by EIA.
[0174] Exposure of Erythrocytes to Reduced Oxygen Tension: Washed
erythrocytes were diluted in Krebs buffer containing bicarbonate
(4.7 KCl, 2.0 mM CaCl.sub.2, 140.5 mM NaCl, 1.2 mM MgSO.sub.4) and
equilibrated for 30 minutes in a tonometer (15% O.sub.2, 5%
CO.sub.2, balance N.sub.2) (normoxia). Erythrocytes were then
exposed to reduced oxygen tension by changing the equilibrating gas
to 5% O.sub.2, 5% CO.sub.2, balance N.sub.2. ATP released from the
erythrocytes as well as pH and blood gas tensions were determined
at 5, 10 and 15 min after exposure to the various gas tensions. In
separate studies, samples were collected into acidified ethanol for
cAMP measurement.
[0175] Determination of Leukocyte and Platelet Contamination of
Erythrocyte Preparations: Smears of the concentrated erythrocyte
preparations were microscopically examined for leukocytes using a
commercial kit (Leukostat). The absence of platelet contamination
of membrane preparations was determined by the absence of the
platelet protein CD-41 as determined by Western analysis.
[0176] Phosphodiesterase Activity Assay: In microcentrifuge tubes,
a 7.5 .mu.L aliquot of erythrocyte membrane preparation was
incubated with 30 .mu.L of cAMP substrate, 7.5 ml of buffer (10 mM
Tris-HCl, pH 7.4), and 15 .mu.L of insulin or various PDE
inhibitors at 37.degree. C. At 0, 5, 10, 20, 30, 45, 60 and 90 min,
samples were centrifuged at 13,000.times.g for 10 min and the
supernatant collected. Aliquots (50 .mu.L) were mixed with C. atrox
venom (15 .mu.L) and allowed to incubate for 30 minutes at
30.degree. C. in a microtiter plate after which 100 .mu.L of Biomol
Green.TM. Reagent was added. After agitation (30 min) for color
development, the intensity at 620 nm was determined.
[0177] Data Analysis: Statistical significance between experiments
was determined using an analysis of variance (ANOVA). In the event
that the F ratio indicates that a change has occurred, a Fisher's
LSD test was done to identify individual differences. Results were
reported as means +/- the standard error from the mean (SEM).
* * * * *