Edward J. Messina

C-peptide induces a concentration-dependent dilation of skeletal muscle arterioles only in presence of insulin

In this study we tested the hypothesis that C-peptide alone or in conjunction with insulin may cause a dilation of skeletal muscle arterioles. First-order arterioles (88 μm) isolated from rat cremaster muscles were pressurized (65 mmHg), equilibrated in a Krebs bicarbonate-buffered solution (pH 7.4), gassed with 10% O2 (balance 5% CO2, 85% N2), and studied in a no-flow state. C-peptide administered at concentrations of 0.3, 1, 3, 10, 100, 300, and 1,000 ng/ml evoked arteriolar dilation that was not concentration dependent. In contrast, the administration of the four lower physiological concentrations of C-peptide to arterioles exposed to a nondilating concentration of insulin evoked a significant concentration-dependent increase in arteriolar diameter from 8.6 to 42.3% above control. The arteriolar dilation to C-peptide in the presence of insulin was completely inhibited by administration of N G-nitro-l-arginine (10-4 M). Responses to ACh and adenosine were not enhanced when these drugs were administered in the presence of insulin. These results indicate that C-peptide has the capacity to evoke arteriolar dilation in skeletal muscle via a nitric oxide-mediated mechanism that appears to be enhanced by an interaction with insulin. Furthermore, the effects of insulin appear to be specific for C-peptide and are not the result of a general enhancement of endothelium-dependent or endothelium-independent dilation.In this study we tested the hypothesis that C-peptide alone or in conjunction with insulin may cause a dilation of skeletal muscle arterioles. First-order arterioles (88 microm) isolated from rat cremaster muscles were pressurized (65 mmHg), equilibrated in a Krebs bicarbonate-buffered solution (pH 7.4), gassed with 10% O2 (balance 5% CO2, 85% N2), and studied in a no-flow state. C-peptide administered at concentrations of 0.3, 1, 3, 10, 100, 300, and 1,000 ng/ml evoked arteriolar dilation that was not concentration dependent. In contrast, the administration of the four lower physiological concentrations of C-peptide to arterioles exposed to a nondilating concentration of insulin evoked a significant concentration-dependent increase in arteriolar diameter from 8.6 to 42.3% above control. The arteriolar dilation to C-peptide in the presence of insulin was completely inhibited by administration of NG-nitro-L-arginine (10(-4) M). Responses to ACh and adenosine were not enhanced when these drugs were administered in the presence of insulin. These results indicate that C-peptide has the capacity to evoke arteriolar dilation in skeletal muscle via a nitric oxide-mediated mechanism that appears to be enhanced by an interaction with insulin. Furthermore, the effects of insulin appear to be specific for C-peptide and are not the result of a general enhancement of endothelium-dependent or endothelium-independent dilation.

Inhibition of NO synthesis or endothelium removal reveals a vasoconstrictor effect of insulin on isolated arterioles

In this study we tested the hypothesis that insulin may differentially affect isolated arterioles from red (RGM) and white gastrocnemius muscles (WGM) because of their differences in function and metabolic profile. We also determined whether the responses of these arterioles are endothelium dependent and mediated by either prostaglandins or nitric oxide (NO). Arterioles were isolated, pressurized to 85 mmHg, equilibrated in Krebs bicarbonate-buffered solution (pH 7.4) gassed with 10% O2 (5% CO2-85% N2), and studied in a no-flow state. Control diameters for first-order arterioles from RGM averaged 77 ± 8 μm and from WGM averaged 77 ± 5 μm. Cumulative dose-response curves to insulin (10 μU/ml, 100 μU/ml, 1 mU/ml, and 10 mU/ml) were obtained in arterioles before and after endothelium removal or administration of either indomethacin (Indo, 10-5 M) or N G-nitro-l-arginine (l-NNA, 10-4 M). Insulin evoked concentration-dependent increases in control diameter of intact RGM and WGM arterioles of 6-26% and 9-28%, respectively. Indo was without any effect on insulin-induced dilation in RGM and WGM arterioles. Insulin-evoked dilation in both RGM and WGM arterioles was completely inhibited and converted to vasoconstriction by endothelium removal and administration of l-NNA. These results indicate that in endothelium-intact arterioles from RGM and WGM, insulin evokes an endothelium-dependent dilation that is equivalent and mediated by NO. In contrast, in the absence of a functional endothelium, insulin evokes arteriolar constriction. The finding that insulin can constrict arterioles, at physiological concentrations, suggests that insulin may play a more significant role in the regulation of vascular tone and total peripheral resistance than previously appreciated.

Microcirculatory effects of prostaglandins E1, E2, and A1 in the rat mesentery and cremaster muscle ☆

Abstract The present investigation was carried out to determine the site(s) of vasomotor activity of Prostaglandins E 1 (PGE 1 ), E 2 (PGE 2 ), and A 1 (PGA 1 ) in the terminal vascular bed of the rat mesentery and cremaster (skeletal) muscle. Studies were also performed to determine the effects of PGE 1 and PGA 1 on arteriolar responsiveness to vasoconstrictor agents. In vivo changes in microvascular diameters in response to topical application of prostaglandins and the vasoconstrictor agents were determined quantitatively with an image shearing television microscope and recording system. In the mesentery, PGE 1 elicited arteriolar dilator responses, while similar doses of PGE 2 and PGA 1 were ineffective. The responses of mesenteric arterioles to PGE 1 were not entirely dose-dependent. In the cremaster muscle, the three prostaglandins studied produced a dose-dependent dilation of all muscular microvessels; the order of potency was PGE 1 > PGE 2 > PGA 1 . Mesenteric and cremasteric arteriolar responses to epinephrine, norepinephrine, angiotensin, and vasopressin were inhibited by PGE 1 , and this inhibition was demonstrable after the dilator action of PGE 1 had terminated. PGA 1 did not alter vascular responsiveness in either tissue. It was concluded that prostaglandins, by virtue of their vasodilator actions and inhibitory effects on microvascular responsiveness, may contribute to local control of blood flow.

Prostacyclin (PGI2) Elicits Reflex Bradycardia in Dogs: Evidence for Vagal Mediation

Summary Administration of the vasode-pressor prostaglandins, PGE1 and PGE2, and of nitroprusside, an agent whose direct vascular activity is similar to prostaglandins, resulted in a reduction in systemic arterial blood pressure accompanied by the expected reflex increase in heart rate. Injection of pros-tacyclin (PGI2) and the prostaglandin precursor, arachidonic acid, into the femoral vein, pulmonary artery, left atrium, and left ventricle of the dog elicited a fall in blood pressure and a concomitant reduction in heart rate in both open- and closed-chest anesthetized dogs. Bilateral vagal section eliminated the bradycardia thus establishing that the PGI2-induced change in heart rate is reflex in origin. Prostacyclin and prostaglandins were provided by Dr. U. Axen and Dr. J. Pike (Upjohn Co.). Dial-urethane was a gift from Ciba-Geigy. We are grateful to Messrs. Arsenio Baez and Maret Panzenbeck for excellent technical assistance.

Prostacyclin (PGI2) mediates hypoxic relaxation of bovine coronary arterial strips.

Bovine coronary arterial strips (BCA) exhibiting spontaneous tone, relax in response to a decrease in the pO2 of the bathing medium. Experiments were performed to determine if prostaglandins (PGs) mediate the oxygen-induced changes in tension. BCA were equilibrated in Krebs-bicarbonate solution at 37 degrees C gassed with 95% O2, 5% CO2 and tension was measured isometrically. When the pO2 of the bathing medium was decreased, BCA exhibited reversible reductions in tension. Switching from 95% O2, 5% CO2 to 95% N2, 5% CO2 (anoxia) elicited an initial relaxation followed by a contraction. In contrast, a change to 5% O2, 5% CO2, 90% N2 (hypoxia) was followed by a sustained relaxation. Re-introduction of O2 to anoxic strips produced a biphasic response: relaxation followed by contraction. Indomethacin or eicosatetraynoic acid (EYA) increased tone and inhibited the relaxation produced by anoxia or hypoxia. Indomethacin or EYA did not inhibit the relaxation of anoxic strips during re-introduction of O2, but did inhibit the contraction partially. Relaxation of arterial strips to arachidonic acid (AA) was similar to relaxation to prostacyclin (PGI2). Anoxia limited the relaxation to AA but not to PGI2. We conclude that PG synthesis contributes to the basal tone and the hypoxia-induced relaxation of BCA. In addition, hypoxia, unless severe, does not prevent the conversion of AA to PGI2.

Modified arteriolar responses to ATP after impairment of endothelium by light-dye techniques in vivo

In this study we investigated whether endothelial cells are involved in the dilation of third-order arterioles (14 to 22 microns) in response to adenosine triphosphate (ATP) in cremaster muscle of pentobarbital-anesthetized rats. Two light/dye (L/D) techniques were employed to achieve selective, local endothelial impairment. One of these techniques utilizes a mercury lamp and sodium fluorescein, the other a Helium-Neon laser and Evans blue dye. L/D treatment (illumination with the appropriate wavelengths of light in the presence of an intravascular dye) of a 20-to 100-microns segment of an arteriole resulted in a complete loss of arteriolar dilation in response to topical administration of acetylcholine (10(-6) M) and arachidonic acid (AA, 10(-5) M). These agents were applied in 100-microl aliquots without interrupting the continuous suffusion with Ringer-gelatin solution and caused a approximately 70% increase in vascular diameter before the L/D intervention. Selectivity of the impairment was assessed by arteriolar responses to the nonendothelium-dependent dilator agents adenosine (10(-5) M) and sodium nitroprusside (2 X 10(-7) M), which elicited the same degree of dilation before and after L/D treatment. Under control conditions ATP (10(-6), 10(-5), and 10(-4) M) elicited dose-dependent increases in arteriolar diameter (from 38 to 74%). After impairment of arteriolar endothelium, dilation in response to all doses of ATP was significantly reduced. Theophylline (30 microM) significantly inhibited arteriolar dilation in response to adenosine (10(-6), 10(-5), and 10(-4) M) but did not affect the responses to various doses of ATP. Moreover, impairment of endothelium enhanced constrictor responses of arterioles to norepinephrine (0.6 X 10(-8) M). These results indicate that arteriolar endothelium of skeletal muscle can mediate or modulate arteriolar responses to various vasoactive agents, suggesting that it has an important role in the regulation of blood flow.

Myogenic responses of isolated adipose tissue arterioles.

Previous in vivo studies indicate that vascular autoregulation does take place in adipose tissue. We tested the hypothesis that adipose tissue arterioles can develop a myogenic response to increases in transmural pressure. Arterioles, isolated from the inguinal fat pad of male Wistar rats, were placed in a microvessel chamber containing a Krebs bicarbonate-buffered solution (pH 7.4) gassed with 10% O2 (5% CO2; 85% N2). Vessels were cannulated and pressurized to 100 mm Hg and studied under no-flow conditions. Control diameters were obtained at 100 mm Hg. Changes in arteriolar diameter were observed and measured by television microscopy and video caliper. Diameters, in response to 20 mm Hg step increases in transmural pressure, were measured before and after removal either of extracellular calcium or of the endothelium, and administration of indomethacin (10(-5) M) or L-NAME (3 x 10(-4) M). Removal of calcium resulted in an increase in control diameter of 81% and completely eliminated the myogenic response. In contrast, administration of indomethacin increased control diameter by 13%. L-NAME significantly enhanced the myogenic response; however, neither endothelium removal nor indomethacin had any significant effect. These results indicate that adipose tissue arterioles are capable of eliciting a myogenic response that could contribute to the regulation of blood flow in vivo. Furthermore, it appears that calcium is essential for the myogenic response and that nitric oxide significantly contributes to the modulation of baseline myogenic tone, as well as the myogenic response.

Depressed arteriolar responsiveness to norepinephrine in streptozotocin-induced diabetes in the rat

We examined the contribution of prostaglandins to altered reactivity to norepinephrine in rat cremaster third order arterioles of streptozotocin (STZ) treated rats and age-matched controls. NE was applied topically to the cremaster muscle of pentobarbital (35 mg/kg) anesthetized rats before and during topical administration of indomethacin (IND: 10 micrograms/ml) four and eight weeks after i.v. injection with of 50 mg/kg STZ (STZ-4W; STZ-8W) or vehicle (C-4W; C-8W), and before and during topical administration of 5,8,11,14 eicosatetraynoic acid (ETYA; 20 micrograms/ml) in STZ-8W and C-8W. Plasma glucose was elevated significantly in STZ-treated rats. Blood pressures and resting arteriolar diameters did not differ. However, vasoconstrictor responses to NE were depressed in STZ-4W and to a greater degree in STZ-8W, IND normalized reactivity to the low doses of NE and partially restored reactivity to the higher doses. ETYA enhanced reactivity to all doses of NE to a greater extent than did IND. These data are consistent with a role for locally produced vasomodulatory arachidonic acid metabolites, including prostaglandins, in the decreased reactivity to NE in diabetic rat cremaster muscle arterioles.

Microcirculatory effects of arachidonic acid and a prostaglandin endoperoxide (PGH2).

Abstract We have examined the vascular effects of arachidonic acid and a prostaglandin endoperoxide, precursors of prostaglandins (PG), in the rat cremaster (skeletal muscle) microcirculation. Five-week-old male, Wistar-strain rats were anesthetized with pentobarbital (30 mg/kg) and the cremaster muscle was prepared for direct in vivo observation and quantitation of changes in vascular diameters in response to the topical application of arachidonic acid (AA), PGH 2 , or PGE 2 . All three agents elicited dose-dependent arteriolar dilator responses. However, threshold doses were different; for AA they were between 0.5 × 10 −7 and 0.5 × 10 −6 M , for PGH 2 between 0.5 × 10 −8 and 0.5 × 10 −7 M , and for PGE 2 between 0.5 × 10 −9 and 0.5 × 10 −8 M . At the high dose studied (0.5 × 10 −3 M ) both AA and PGH 2 required approximately 60 sec for the development of a maximum dilator response, whereas PGE 2 required only about 30 sec. Indomethacin, an inhibitor of prostaglandin synthesis, did not significantly alter control arteriolar diameters but inhibited completely the responses to AA. We conclude that the rat cremaster skeletal muscle microcirculatory compartment does possess the necessary enzymatic machinery for the conversion of AA and PGH 2 into vasodilator prostaglandin metabolites.

General considerations for evaluation of cutaneous and skeletal muscle blood flow.

The purpose of this paper is to review briefly some of the anatomic, physiologic and pharmacologic features of the two major vascular compartments of the lower extremity, namely the cutaneous and skeletal muscle circulations. Both skin and skeletal muscle possess the same series elements of the vascular tree, that is, arteries, arterioles, capillaries, post-capillary venules and veins, found in other vascular beds. Yet, each is unique and unlike any other vascular bed in both vascular design and function. Attempts, therefore, to evaluate the sufficiency of a regional circulation must have as its basis (1)