Hindlimb Vascular Bed Research Articles

Effects of the Rho/Rho-Kinase Pathway on Perfusion Pressure in the Isolated-Perfused Rat Hind Limb Vascular Bed.

Background: Rho/ROCK signaling has been demonstrated to be involved in the vascular reactivity of many arterial networks. However, RhoA expression and the contribution of Rho/ROCK pathway to the control of perfusion pressure have not been investigated in the rat hind limb vascular bed as a skeletal muscle vascular network. Aims: To investigate the contribution of the Rho/ROCK pathway in the control of perfusion pressure in the isolated-perfused rat hind limb vascular bed.Study Design: Animal experimentation.Methods: Two Rho inhibitors (atorvastatin and C3 exoenzyme) and ROCK inhibitors (Y-27632 and fasudil) were tested on the phenylephrine-elevated perfusion pressure in the isolated-perfused rat hind limb vascular bed. Furthermore, we sought the expression of RhoA protein in the femoral, popliteal and saphenous arteries as well as quadriceps and gastrocnemius muscles by Western blotting.Results: The ROCK inhibitors Y-27632 and fasudil (both 10−8 to 10−5 M) induced substantial vasodilatations. The maximum vasodilatations induced by Y-27632 and fasudil (both at 10−5 M) were 84.0 ± 6.9% and 76.9 ± 6.9%, respectively (P = .091). Y-27632 was not more potent than fasudil, as the EC50 values for Y-27632 and fasudil were 0.7 ± 2.1 μM and 2.5 ± 2.4 μM, respectively (P = .177). Atorvastatin (10−7 to 10−4 M) and C3 exoenzyme (3 × 10−8 M) also produced vasodilatation (maximum vasodilatation; 20.3 ± 1.7% and 13.7 ± 3.6%, respectively). The EC50 value for atorvastatin was 94.9 ± 1.2 μM. The western blot analysis showed that the femoral, saphenous, and popliteal arteries, as well as the gastrocnemius and quadriceps muscles, express RhoA protein.Conclusion: The Rho/ROCK pathway contributes significantly to the control of perfusion pressure in the rat hind limb vascular bed.

Physiological Function of Blood Vessels from Uncontrolled Donation after Circulatory Death in Pigs

Donation after circulatory death (DCD) has become increasingly important in kidney transplantation. Challenges, including early graft loss and delayed graft function, remain. Little is known regarding vascular properties. The aim was to examine effects of DCD on renal artery function in a novel pig extended DCD model, extending the time from circulatory arrest to retrieval of the organ to more than 4 hours, making it more feasible to recruit more organs from DCD. Renal and iliac arteries were obtained from animals immediately after euthanasia (fresh) and 4.5hr after DCD. Vessels were also analyzed after 10hr at 4°C in physiological salt solution (stored). Preparations were mounted for isometric force recording in vitro and examined at optimal stretch for active tension. Iliac arteries were more sensitive to noradrenaline compared to renal artery, possibly reflecting differences in sympathetic control of perfusion in renal and hind limb vascular beds. Active tension (depolarization, noradrenaline activation) was significantly lower in DCD renal arteries compared to fresh vessels. Stored renal arteries were also weaker, but to a lower extent, whereas iliac artery was only marginally weakened in uDCD and unaffected by storage. For both vessel types, DCD caused a significant attenuation of endothelial-mediated relaxation. Kidneys from uDCD donors performed poorly after transplantation, as expected, with a somewhat better result if the donor was given the standard protocol, including cooling after two hours. In conclusion, DCD introduces changes both in contractile function and endothelium-mediated relaxation of renal arteries. Effects were more pronounced than in iliac artery, suggesting that DCD could be one factor affecting the functional circulation of the donor kidney after transplantation. Further studies of uDCD subject to reconditioning before transplantation is underway to study the reversibility of changes to the endothelial function after our uDCD protocol.

Orchidectomy attenuates high-salt diet-induced increases in blood pressure, renovascular resistance, and hind limb vascular dysfunction: role of testosterone.

Sex hormone-dependent vascular reactivity is an underlying factor contributing to sex differences in salt-dependent hypertension. This study evaluated the role of androgens (testosterone) in high salt-induced increase in blood pressure (BP) and altered vascular reactivity in renal blood flow and perfused hind limb preparation. Weanling male rats (8weeks old, 180-200g) were bilaterally orchidectomised or sham operated with or without testosterone replacement (Sustanon 250, 10mg/kg intramuscularly once in 3weeks) and placed on a normal (0.3%) or high (4.0%) NaCl diet for 6weeks. The high-salt diet (HSD) increased arterial BP, renal vascular resistance (RVR) and positive fluid balance (FB). These changes were accompanied by decreased plasma nitric oxide levels. The increased BP, RVR and FB observed in the rats fed a HSD were reversed by orchidectomy while testosterone replacement prevented the reversal. Phenylephrine (PE)-induced increased vascular resistance in the perfused hind limb vascular bed was enhanced by HSD, the enhanced vascular resistance was prevented by orchidectomy and testosterone replacement reversed orchidectomy effect. Vasorelaxation responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were impaired in HSD groups, orchidectomy attenuated the impairment, while testosterone replacement prevented the orchidectomy attenuation. These data suggested that eNOS-dependent and independently-mediated pathways were equally affected by HSD in vascular function impairment and this effect is testosterone-dependent in male Sprague-Dawley rats.

Respiratory, metabolic and cardiac functions are altered by disinhibition of subregions of the medial prefrontal cortex

The prefrontal cortex (PFC) is referred to as the visceral motor cortex; however, little is known about whether this region influences respiratory or metabolic outflows. The aim of this study was to describe simultaneous changes in respiratory, metabolic and cardiovascular functions evoked by disinhibition of the medial PFC (mPFC) and adjacent lateral septal nucleus (LSN). In urethane-anaesthetized rats, bicuculline methiodide was microinjected (2 mm; GABA-A receptor antagonist) into 90 sites in the mPFC at 0.72-4.00 mm from bregma. Phrenic nerve amplitude and frequency, arterial pressure, heart rate, splanchnic and lumbar sympathetic nerve activities (SNA), expired CO2, and core and brown adipose tissue temperatures were measured. Novel findings included disturbances to respiratory rhythm evoked from all subregions of the mPFC. Injections into the cingulate cortex evoked reductions in central respiratory function exclusively, whereas in ventral sites, particularly the infralimbic region, increases in respiratory drive and frequency, and metabolic and cardiac outflows were evoked. Disinhibition of sites in surrounding regions revealed that the LSN could evoke cardiovascular changes accompanied by distinct oscillations in SNA, as well as increases in respiratory amplitude. We show that activation of neurons within the mPFC and LSN influence respiratory, metabolic and cardiac outflows in a site-dependent manner. This study has implications with respect to the altered PFC neuronal activity seen in stress-related and mental health disorders, and suggests how basic physiological systems may be affected.

Analysis of Vancomycin in the Hindlimb Vascular Bed of the Rat

Analysis of Vancomycin in the Hindlimb Vascular Bed of the Rat

Sexual dimorphism of vascular mechanics in rats

The purpose of this study was to test the hypotheses that male and female rats would exhibit dimorphic mechanical properties of the hindlimb vasculature and that these differences would persist in hypertension. Vascular resistance (R), compliance (C), and tissue viscoelasticity (K) of the hindlimb vascular bed were quantified from concurrent femoral artery blood flow and pressure waveforms (lumped Windkessel model) in age‐matched Wistar‐Kyoto (WKY) and spontaneously hypertensive rats (SHR) male (n=10 each) and female (n=6 each). Female WKY rats displayed greater R (15.4±5.7 vs 7.7±3.3mmHg/mL/min; P<0.05) and K (3.4±2.4 vs 1.1±2.7mmHg/mL/min; P<0.05) with lower C (3.4E‐3±0.8E‐3 vs 4.9E‐3±0.9E‐3mL/mmHg/g; P<0.05) compared to males, respectively. Similarly, female SHRs had higher R (31.5±8.5 vs 17.6±7.8mmHg/mL/min; P<0.05) and K (7.3±2.5 vs 2.9±1.0mmHg/mL/min; P<0.05) with lower C (4.8E‐3±2.0E‐3 vs 6.8E‐3±3.5E‐3mL/mmHg/g; P<0.05) than their male counterparts. Main effects of strain and sex were observed for each of R, K and C variables (all P<0.05). Therefore, sex differences are apparent in the indices of steady state (R) and oscillatory (K and C) hindlimb vascular mechanics and these persist in hypertension.Supported by CIHR.

Analysis of Vancomycin in the Hindlimb Vascular Bed of the Rat: ERRATUM

Analysis of Vancomycin in the Hindlimb Vascular Bed of the Rat: ERRATUM

Responses to arachidonic acid and other dilator agonists and their modification by inhibition of prostaglandin synthesis in the canine hindlimb.

The influence of indomethacin, in doses that completely inhibit the response to arachidonic acid, has been examined on canine hindlimb vascular responses to intra-arterial administration of bradykinin, histamine, nitroglycerin, isoprenaline and papaverine. The hindlimb was perfused at constant flow. Dose-response curves to intra-arterial administration of arachidonic acid (12 to 200 microgram kg-1), bradykinin (0.4 to 100 ng kg-1), histamine (1.8 to 120 ng kg-1), nitroglycerin (15 to 100 ng kg-1), isoprenaline (12 to 100 ng kg-1) and papaverine (1.2 to 160 microgram kg-1) (all n = 4) were compared before and 30 min after indomethacin (5 mg kg-1 i.v.). All the drugs produced dose-related decreases in hindlimb perfusion pressure. After indomethacin, responses for all dilator agonists except arachidonic acid, were significantly greater than control (P less than 0.05), both in terms of absolute (mmHg) or percent change. Dose-response curves after indomethacin had a left upward shift compared with control. Arachidonic acid responses were completely blocked by indomethacin. These findings suggest that indomethacin produces a non-specific increase in responsiveness of the hindlimb vascular bed to dilator substances, except arachidonic acid. The data presented do not support the hypothesis that the peripheral vasodilatation produced by bradykinin, nitroglycerin and histamine could be mediated by endogenous prostaglandin release.

Chronic Angiotensin II Infusion Causes Differential Responses in Regional Sympathetic Nerve Activity in Rats

Angiotensin II (AngII)-induced hypertension in experimental animals has been proposed to be attributed in part to activation of the sympathetic nervous system. This sympathetic activation appears to be accentuated in animals consuming a high-salt diet (AngII-salt hypertension). However, accurate quantification of sympathetic activity is difficult, and controversy remains. It is particularly important to ask which are the critical vascular beds targeted by increased sympathetic nerve activity (SNA) in AngII-salt hypertension. To address this issue, mean arterial pressure and renal SNA or lumbar SNA were continuously recorded during a 5-day control period, 11 days of AngII (150 ng/kg per minute, SC), and a 5-day recovery period in conscious rats on a high-salt (2% NaCl) diet. Although mean arterial pressure reached a new steady-state level of 30 to 35 mm Hg above control levels by the end of the AngII period, renal SNA decreased by 40% during the first 7 days of AngII and then returned toward control levels by day 10 of AngII. In contrast, lumbar SNA remained at control levels throughout the AngII period. In another experiment we measured hindlimb norepinephrine spillover in conscious rats on normal (0.4%) or high- (2.0%) salt diets before and during 14 days of AngII administration. AngII had no significant affect on hindlimb norepinephrine spillover in either group. We conclude that chronic AngII modulates renal and lumbar SNAs differentially in rats consuming a high-salt diet and that AngII-salt hypertension in the rat is not caused by increased SNA to the renal or hindlimb vascular beds.

Alteration of endothelium-mediated vasodilator response in the rat hindlimb vasculature consecutive to chronic hypoxic stress: NO and EDHF involvement

The previously documented impairment of hindlimb blood flow consecutive to chronic hypoxia might be related to endothelial vasomotor dysfunction. The aim of this study was to assess in-vivo the effect of chronic hypoxic stress on endothelium-mediated vasodilator response of hindlimb vascular bed, especially as regards to endothelium-derived hyperpolarizing factor (EDHF) and nitric oxide (NO) pathway contribution. Dark Agouti rats were randomly assigned to live at barometric pressure ≈ 760 mmHg (N rats) or ≈ 550 mmHg (CH rats). Under anesthesia, catheters were placed in the carotid artery for arterial pressure measurement, and in the saphenous vein and iliac artery for drug delivery. Hindlimb blood flow (HBF) was measured by transit-time ultrasound flowmetry, at baseline and during endothelium-dependent vasodilator response induced by intra-arterial injection of acetylcholine (0.75 ng and 7.5 ng) with and without specific blockers of NOS (L-NAME) and EDHF (Charybdotoxin + Apamin). HBF and hindlimb vascular conductance changes in response to ACh infusion were significantly lower in CH than in N rats. The mechanisms responsible for this blunted response involved impairment in both NO pathway and EDHF. The chronic hypoxia-induced alteration of NO pathway was mainly related to the bioavailability of its substrate l-Arginine, since the infusion of l-Arginine restored the endothelial response to ACh in CH rats to the level of N rats. These results demonstrate that the impairment in endothelium-mediated vasodilator response of the hindlimb vascular tree induced by chronic hypoxic stress involves both NO and EDHF.

Analysis of Vasodilator Responses to Peroxynitrite in the Hindlimb Vascular Bed of the Cat

The free radical peroxynitrite (ONOO-) is formed in biological systems from the reaction of nitric oxide (NO) with superoxide (O2-) and can react with protein and nonprotein thiol groups to produce tissue injury. However, these pathologic actions of (ONOO-) may have been overemphasized, in that (ONOO-) has vasorelaxant properties through activation of soluble guanylate cyclase; inhibits leukocyte-endothelial cell interactions; and reduces ischemia-reperfusion injury in the heart, lung, and liver. It has been reported that tolerance develops to the vasodilator actions of (ONOO-) and that (ONOO-) impairs vascular function. However, little, if anything, is known about responses to (ONOO-) in the hindlimb circulation of the cat. To better understand the effects of (ONOO-) on responses to vasoactive agonists and the mechanism by which (ONOO-) induces vasodilation, the effects of short-term exposure to (ONOO-) were investigated under constant-flow conditions in the hindlimb vascular bed of the cat. In these studies, direct intraarterial injections of (ONOO-) produced dose-dependent decreases in hindquarters perfusion pressure. The vasodilator responses to (ONOO-) were rapid in onset, were short in duration, and could be repeated without exhibiting tachyphylaxis. Vasodilator responses to (ONOO-) were not changed in the presence of inhibitors of nitric-oxide synthase, cyclooxygenase, or K+-ATP (adenosine triphosphate-sensitive potassium) channels. Furthermore, responses to (ONOO-) were enhanced in duration by the type 5-cGMP (cyclic guanosine monophosphate) phosphodiesterase inhibitor zaprinast, whereas rolipram, a type 4-cGMP phosphodiesterase inhibitor, was without effect. Repeated administration of (ONOO-) had no significant effect on responses to vasoconstrictor or to vasodilator agents including acetylcholine. These results show that (ONOO-) has significant vasodilator activity in the hindlimb vascular bed of the cat and suggest that the response is mediated by a cGMP- dependent mechanism. The results of experiments with repeated injections of (ONOO-) indicate that (ONOO-) does not impair vasoconstrictor and endothelium-dependent or endothelium-independent vasodilator responses. Furthermore, tolerance did not develop with repeated short-term exposure to (ONOO-). Moreover, the results of experiments with inhibitors suggest that responses to (ONOO-) are not dependent on K-ATP (adenosine triphosphate-sensitive potassium) channel activation, increased NOS activity, or the formation of products in the cyclooxygenase pathway. The results of these studies are consistent with the hypothesis that (ONOO-) is rapidly converted in the hindlimb circulation to a substance that has the properties of an NO donor. These studies suggest that under physiologic conditions, the cytotoxic effects of (ONOO-) on a short-term basis may have been overemphasized.

Hemodynamic Responses Elicited By γ 2-MSH or Blood Replacement in Hemorrhaged Rats

Systemic injections of compounds such as gamma(2)-melanocyte-stimulating hormone (gamma(2)-MSH), which increase sympathetic neurogenic vasoconstriction, may be beneficial in treating hemorrhage-induced hypotension. This study characterized (1) the hemodynamic responses elicited by systemic injections of gamma(2)-MSH in pentobarbital-anesthetized hemorrhaged rats, and (2) the hemodynamic responses elicited by the replacement of withdrawn blood in these rats. Controlled hemorrhage (4.8 +/- 0.3 mL/rat at 1.5 mL/min) resulted in a pronounced and sustained fall in mean arterial blood pressure (MAP). The fall in MAP was associated with a reduction in heart rate (HR) and hindquarter (HQR) vascular resistance but no changes in mesenteric (MR) or renal (RR) vascular resistances. Systemic injections of gamma(2)-MSH (10-40 microg/kg, i.v.) produced dose-dependent increases in HR, MAP, and vascular resistances of 20 to 60 s in duration in the hemorrhaged rats. In contrast, injection of the withdrawn blood produced an immediate and sustained increase in MAP, which was associated with a pronounced vasodilation in the hindquarter bed but no changes in MR or RR. These findings suggest that although gamma(2)-MSH elicits pressor and vasoconstrictor responses in hemorrhaged rats, the bolus injection of this peptide may not in itself be an effective strategy for the sustained restoration of MAP in these rats. Moreover, although blood replacement effectively restores MAP via increases in cardiac output rather than total peripheral resistance, it appears that this manipulation produces an active vasodilation in the hindquarter bed. The possibility that this vasodilation involves a sympathetic neurogenic vasodilator system, which innervates the hindlimb vascular bed but not mesenteric or renal vascular beds, will be discussed.

Lack of dilator effect of leptin in the hindlimb vascular bed of conscious rats

Leptin acts in the hypothalamus to decrease appetite and increase sympathetic nerve activity. Some in vitro studies have suggested that leptin may possess vasodilator actions that oppose sympathetically mediated vasoconstriction. We tested whether leptin is a vasodilator of the hindlimb vascular bed in vivo, and also whether it alters vasodilation to acetylcholine or sodium nitroprusside. In conscious instrumented Sprague–Dawley rats we measured iliac blood flow responses to local arterial infusion of acetylcholine (0.1 and 1 μg/min) and sodium nitroprusside (1 and 10 μg/min) before and after 2 h of intra-arterial leptin (at doses of 0.1 or 1 mg/kg, n=10 each) or vehicle (n=10). Leptin infusion over 2 h did not change mean arterial pressure, heart rate or iliac flow and conductance. In addition, leptin did not significantly alter the vasodilation (increase in the conductance ratio between infused and non-infused arteries) induced by acetylcholine, whereas pre-treatment with a nitric oxide synthesis inhibitor, NG-nitro-l-arginine methyl ester, significantly attenuated the vasodilation to acetylcholine. Leptin also did not affect the vasodilation to sodium nitroprusside. Lack of effect of leptin on vasodilator function could not be due to the activation of the sympathetic nervous system because lumbar sympathoactivation was obtained only 4 h after the iliac infusion of 1 mg/kg/h of leptin. These findings are in accordance with our previous results and argue against a meaningful direct dilator action of leptin on resistance vessels in vivo.

Effect of binge cocaine treatment on hindlimb vascular function

Chronic cocaine abuse is known to cause endothelial dysfunction and atherosclerosis. The present study investigated the effect of binge cocaine treatment, a model for chronic cocaine abuse, on the blood flow responses to the adrenergic agonists norepinephrine, phenylephrine and isoproterenol, the endothelium-dependent vasodilator acetylcholine, and the endothelium independent vasodilator sodium nitroprusside (SNP) in the hindlimb vascular bed of male Sprague Dawley rats. Rats received either single binge or double binge treatment. Each binge treatment consisted of three doses of cocaine (30 mg kg(-1) i.p.) for 3 days. For double binge treatment, there was a 4 day recovery period between the binges. At the end of the treatment the rats were anesthetized and agonists were administered into the right hindlimb circulation through a catheter in the left iliac artery and blood flow responses were measured with a flow probe around the right iliac artery. Rats receiving double cocaine binges showed a significant decrease in the magnitude and duration of the blood flow response to norepinephrine and a decrease in the duration of the blood flow response to phenylephrine, isoproterenol and acetylcholine when compared with saline controls. The blood flow response to SNP was not changed. Total plasma nitrate-nitrite levels were significantly reduced and big endothelin levels were significantly increased in rats receiving double cocaine binges. This study demonstrates that binge cocaine treatment can alter endothelial function, while not changing smooth muscle function, and impairs the adrenergic pathway.

Responses to bradykinin are mediated by NO-independent mechanisms in the rat hindlimb vascular bed

The vasodilator response to bradykinin (BK) appears to be mediated by a number of different endothelium-derived relaxing factors (EDRFs). The EDRFs mediating the response depend on the species and vascular bed studied. The mechanism by which BK dilates the hindlimb vascular bed was investigated in the anesthetized rat. BK produced dose-dependent increases hindlimb blood flow. The NOS antagonist L-NAME had little effect on the magnitude of the increase in flow when baseline hemodynamic parameters were corrected by an NO donor infusion. However, the duration of the response was slightly shortened by L-NAME. Charybdotoxin (Chtx) and apamin nearly abolished the L-NAME resistant component of the hindlimb vasodilator response to BK, but did not affect the hindlimb vasodilator response to the sodium nitroprusside (SNP). The cyclooxygenase inhibitor meclofenamate and the K +-ATP channel blocker U37883A, in the presence of L-NAME, did not alter the vasodilator response to BK. These results suggest that a significant portion of the hindlimb vasodilator response to BK is mediated by the activation of K Ca channels, and independent of NO synthesis, cyclooxygenase products, and activation of K +-ATP channels. The present data suggest that the mechanisms mediating the vasodilator response to BK in the hindlimb vascular bed of the rat are complex, consisting of a Chtx and apamin sensitive, L-NAME resistant phase and a minor phase mediated by NO. In contrast, NO accounts for about half of the hindlimb vasodilator response to acetylcholine (ACh), with the other half of the response mediated by a Chtx and apamin sensitive mechanism. Additionally, the present results demonstrate that the NO donor infusion technique is able to compensate for the loss of basal NO production following inhibition of NOS, and to restore hemodynamic parameters to pre-L-NAME levels, making it a useful technique for the investigation of the role of NO in mediating vascular responses.

Role of potassium channels in the nitric oxide-independent vasodilator response to acetylcholine

Stimulation of vascular endothelial muscarinic receptors by acetylcholine (ACh) leads to the formation of an endothelium-derived relaxing factor (EDRF), which is generally accepted to be nitric oxide (NO). Recent evidence, however, suggests that NO may be only one of several EDRFs mediating the vasodilator response to ACh. Since this NO-independent vasodilator response to ACh has been hypothesized to be dependent upon K + channel activation, the current study was undertaken to investigate the role of K + channels in mediating the hindlimb vasodilator responses to ACh in vivo. Additionally, since variations in vascular tone can complicate the analysis of responses, the level of vascular tone was maintained at a similar level throughout the study so that responses could be compared directly. The results of the present study demonstrate that the vasodilator response to ACh possesses a significant component that is independent of NO production. The K Ca channel blockers charybdotoxin and apamin, but not K +-ATP channel blocker U37883A or the COX antagonist meclofenamate, attenuated the NO-independent component of the vasodilator response to ACh. This suggests that K Ca channels, but not K +-ATP channels or COX products, are involved in mediating the l-NAME resistant response to ACh. Further, the inhibition of the ACh vasodilator response by the K +-ATP opener BRL55834 suggests that the response is dependent upon membrane hyperpolarization. These data suggest that the mechanism mediating ACh responses in the hindlimb vascular bed of the rat are complex and may involve several signaling pathways.

Differential responses to ATPγS in the mesenteric and hindlimb vascular bed of the cat

The mechanism by which the purinergic agonist adenosine 5′- O-(3 thiotriphosphate) (ATPγS) decreases vascular resistance was investigated in the mesenteric and hindlimb vascular beds of the cat. Injections of ATPγS into the hindlimb perfusion circuit elicited dose-dependent decreases in perfusion pressure while injections into the mesenteric circuit produced a biphasic response with an initial vasopressor response followed by a vasodepressor response. In the mesenteric vascular bed the pressor response to ATPγS was blocked by a P2X 1 receptor antagonist. Also an inhibitor of nitric oxide synthase enhanced the vasoconstrictive responses to ATPγS. However, the vasodepressor response in the mesenteric bed was not altered by the adminstration of an alpha adrenergic receptor antagonist, a cyclooxygenase inhibitor, a P2Y 1 receptor antagonist, or a K + ATP channel blocking agent. These data suggest that the vasopressor response to ATPγS in the mesenteric vascular bed of the cat is mediated via P2X 1 receptor activation. The differential responses to ATPγS in the hindlimb and mesentery suggest differences in purinergic receptor distribution in the vascular system of the cat. In addition, the results suggest that prostaglandin synthesis, P2Y 1 receptor activation, alpha receptor inhibition, and K + ATP channels activation play little to no role in mediating the vascular response to ATPγS in the mesentery of the cat.

In vivo Adenoviral Gene Transfer of CGRP and Related Peptides to the Rat and Mouse

Calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM) play an important role in maintaining low pulmonary vascular resistance (PVR) and may be involved in modulating the pulmonary vascular response to chronic hypoxia. Adenoviral vectors encoding prepro-CGRP (AdRSVCGRP) and ADM (AdRSVADM) were used to examine the effects of in vivo gene transfer of the peptides to the lung on increases in PVR, right ventricular mass, and pulmonary vascular remodelling that occurs in chronic hypoxia in the mouse. Intratracheal administration of AdRSVCGRP or AdRSVADM, followed by 16 days of chronic hypoxia (FIO2 0.10), increased lung CGRP and ADM levels, respectively. The increase in pulmonary arterial pressure (PAP), PVR, right ventricular mass, and pulmonary vascular remodeling in response to chronic hypoxia was attenuated in animals overexpressing prepro-CGRP or ADM whereas systemic arterial pressure was not altered. In chronically hypoxic mice, increases in PAP in response to i.v. injections of angiotensin II and endothelin-1 were attenuated following in vivo gene transfer. These data show that in vivo transfer of the CGRP or ADM gene to the lung attenuates the increase in PVR and right ventricular mass, pulmonary vascular remodeling, and responsiveness in chronically hypoxic mice with little effect on the systemic circulation. In order to study the roles of RAMP 1, 2, and 3 in mediating responses to CGRP and related peptides in the pulmonary and systemic vascular beds, gene transfer of adenovirus vectors encoding CRLR and RAMP 1, 2, or 3 were delivered to the lung and/or hindlimb of the mouse. Transfection of CRLR and RAMP 2 to the pulmonary and hindlimb vascular beds of the mouse resulted in enhanced vasodilator responses to ADM, but not CGRP.

Analysis of the hemodynamic effects of heparinase I and protamine sulfate in the systemic and hindlimb vascular beds of the male rat.

The effects of heparinase I and protamine sulfate on the mean arterial pressure and hindlimb perfusion pressure in the male rat were studied. With institutional approval, 21 male Sprague-Dawley rats were anesthetized, and the carotid artery and abdominal aorta were cannulated by cutdown. To isolate the hindlimb, a semi-closed peristaltic perfusion circuit was used. Heparinase I or protamine sulfate was injected into the hindlimb vascular bed, and changes in mean arterial pressure and hindlimb perfusion pressure were recorded. Analysis of variance with a post hoc Scheffe's test was used for statistical analysis, and a P value less than.05 was considered significant. Increasing doses of heparinase I caused a small but significant decrease in mean arterial pressure only at the two highest doses. At all doses, hindlimb perfusion pressure was significantly less than the baseline value and than the value with saline administration at 1 minute. At the clinically applicable doses of heparinase I (0.625 and 1.25 IU/kg), the decrease in hindlimb perfusion pressure was less than 7. At the next two higher doses, the change was less than 15%. The vehicle of heparinase caused a significant decrease in mean arterial pressure (from -15% to -30%) and hindlimb perfusion pressure (from -10% to -20%). Increasing doses of protamine sulfate caused an increase in hindlimb perfusion pressure from baseline, including a 58% change with the 10-mg/kg dose. There was a transient decrease in mean arterial pressure, which peaked 4 to 5 minutes after injection, to a 21% decrease from baseline with the 5- and 10-mg/kg doses. Heparinase I caused vasodilation in the hindlimb and decreased mean arterial pressure only at supraclinical doses. Protamine sulfate caused a significant dose-dependent increase in hindlimb vascular resistance and a transitory decrease in mean arterial pressure.

Gene transfer of endothelial nitric oxide isoform decreases rat hindlimb vascular resistance in vivo.

The objective of this study was to design a methodology of gene transfer into a resistance vascular bed and to show if such a method can be used to examine the physiological function of a given gene product in vivo. We developed such a method and validated it by defining the role in vivo of endothelial nitric oxide synthase (eNOS). In a constant flow perfused rat hindlimb, gene transfer to the vascular endothelium was accomplished by incubating a "first-generation" serotype 5, replication-deficient, adenoviral vector (1.2 X 10(9) plaque-forming units/ml) containing cDNA encoding either the eNOS or the beta-galactosidase (beta-Gal) gene in the hindlimb vasculature for 30 min. Five days after infection, immunohistochemical staining for eNOS localized recombinant gene expression to vascular endothelial cells and eNOS protein levels were increased fourfold (11.9 +/- 6.6 vs. 2.9 +/- 1.3 intensity units/microg protein, n = 4, p < 0.05). Perfusion pressures were measured at different flow rates (10-50 ml/min). In addition, basal and acetylcholine (ACh)-stimulated vascular resistance (VR) in phenylephrine (PE)-precontracted (100 microM) hindlimb was measured at constant flow. There were flow-dependent increases (p < 0.05) in perfusion pressure. Overexpression of eNOS shifted the pressure-flow curve downward and administration of N(G)-nitro-L-arginine methyl ester (L-NAME) shifted the curve upward. Compared with beta-Gal-transfected rats, PE-induced VR decreased (p < 0.05) in eNOS-transfected rats (100 +/- 27 vs. 164 +/- 49 mmHg, n = 5). Addition of 100 microM L-NAME increased (p < 0.05) PE-induced VR in both eNOS-transfected and control rats (145 +/- 50 and 232 +/- 38 mmHg, n = 5, p < 0.05), respectively, which was partially abolished by L-arginine pretreatment. ACh-induced vasorelaxation was increased 45% (p < 0.05) in eNOS-transfected hindlimbs. L-NAME decreased (p < 0.05) ACh-induced vasorelaxation by 58% in eNOS-transfected hindlimbs versus 25% in beta-Gal-transfected hindlimbs (p < 0.05). We used this gene transfer method to examine the physiological function of a gene product in vivo and showed that (1) the flow-pressure relationship in the hindlimb vascular bed is NO dependent and (2) the eNOS enzyme modulates NO-mediated vasorelaxation in the rat hindlimb resistance arteries in vivo.