Distributions of microvascular pressure in skeletal muscle of one-kidney, one clip, two-kidney, one clip, and deoxycorticosterone-salt hypertensive rats.

Studies were performed on the cremaster skeletal muscle in rats to investigate the microvascular changes that are associated with established one-kidney, one clip (1K1C) and two-kidney, one clip (2K1C) Goldblatt hypertension and with deoxycorticosterone (DOC)-salt hypertension. Rats were anesthetized with urethane and chloralose; and cremaster muscles with intact circulation and innervation were suspended in a controlled Krebs bath. Microvascular pressures and vessel diameters were measured at three consecutive arteriolar (A) and venular (V) branch levels. Arteriolar diameters (means +/- SEM) in normotensive (NT) rats were 119 +/- 7, 86 +/- 5, and 31 +/- 3 micron respectively for 1A, 2A, and 3A arterioles; and venule diameters were 218 +/- 12, 141 +/- 15, and 53 +/- 7 micron respectively for 1V, 2V, and 3V venules. As compared to NT rats, there was a selective decrease in lumen size (percent reduction from control) for 1A and 2A (23% to 38%) in 1K1C and 2K1C rats and for 1A, 2A, and 3A (42% to 44%) in DOC rats. Venule diameters were not significantly different between normotensive and hypertensive animals at any branch level. Femoral artery pressures were significantly elevated (greater than or equal to 43%) in all three forms of hypertension; however, this increase in pressure was not proportionally transmitted throughout the microcirculation. This was evidenced by normal pressure in 3A arterioles and in all venules for 1K1C and 2K1C rats and by normal pressures in 3V and larger venules for DOC rats. Our findings indicate that elevated arterial pressure in chronic renal hypertension is not transmitted uniformly across all microvascular segments.(ABSTRACT TRUNCATED AT 250 WORDS)

Progressive microvascular alterations with the development of renovascular hypertension.

Norepinephrine-induced changes in diameters of first- (1A), second- (2A), and third-order (3A) arterioles in the exposed cremaster muscles of normotensive and renovascular hypertensive rats were quantitated via television microscopy. By 2 weeks following the surgery to induce hypertension, we found that 3A sensitivity to norepinephrine had increased and the 1As had chronically constricted. By 4 weeks, the constriction had progressed to include both 1A and 2A. Sensitivity was no longer increased in 3As and, in fact, sensitivity had decreased in 1As and 2As. The 1As and 2As could not be dilated with isoproterenol or nitroprusside; thus, the vessels appeared to have undergone a structural alteration. We suggest from these results that the early increased 3A sensitivity contributes to the initial development of hypertension. The larger arterioles then constrict to protect the downstream vessels from increased luminal pressure. As the hypertension develops, the constriction progresses to smaller arterioles in an attempt to maintain normal pressure in the capillaries (site of water exchange). The constricted arterioles contribute to increased total peripheral resistance, and with the constriction, there occurs a general decrease in vessel responsiveness.

Decreased influence of nitric oxide on deoxycorticosterone acetate (DOCA)-salt hypertension.

The response to exogenous administration of acetylcholine and the effects of nitric oxide (NO) synthesis blockade were assessed for small arterioles in the cremaster muscle of deoxycorticosterone acetate (DOCA)-salt hypertensive and normotensive rats using intravital microscopy. The NO synthesis inhibitor N omega-L-nitro-arginine methyl ester (L-NAME) produced significantly less constriction in the arterioles of DOCA-salt hypertensive rats. Exposure to increasing concentrations of acetylcholine (10(-10) to 10(-5) mol/L) or sodium nitroprusside (10(-10) to 10(-5) mol/L) produced similar arteriolar dilation in both groups. These results suggest that attenuated basal release of NO by arterioles may play a role in the development of increased peripheral resistance observed in DOCA-salt hypertension.

Microvascular responses to endothelin in deoxycorticosterone acetate-salt hypertensive rats.

The objective of the present study was to determine if endothelin-1 played a role in the elevated peripheral resistance in deoxycorticosterone-acetate (DOCA)-salt hypertension. Radioimmunoassay showed that the concentration of endothelin-1 was higher in thoracic aorta of the DOCA-salt group than that of the control normotensive (CN) group. Responses of arterioles in the rat cremaster to endothelin-1 were also observed using in vivo closed circuit television microscopy. Microvascular sensitivity to endothelin-1 was decreased in the DOCA-salt group. In the presence of an endothelin type A (ET-A) receptor antagonist, low concentrations of endothelin-1 induced a significant vasoconstriction in the DOCA-salt group. In the presence of endothelin type B (ET-B) receptor antagonist, microvascular responses to endothelin-1 were attenuated in the DOCA-salt group. These results indicated that the increased tissue level of endothelin-1 may decrease the ET-A receptor-mediated vascular response to endothelin-1. However, the ET-B receptor-mediated vasoconstriction is potentiated during DOCA-salt hypertension.

In vivo platelet thrombus formation in microvessels of spontaneously hypertensive rats.

Sustained high blood pressure causes functional changes in both vascular endothelial cells and platelets. Therefore, we hypothesized that in vivo platelet thrombus formation would be increased in the cremaster muscle microvessels of rats during genetic hypertension. Experiments were carried out on spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) at 12 weeks of age. Fluorescein isothiocyanate tagged to bovine serum albumin (FITC-BSA) was injected intraarterially and 450 to 490 nm light was used to activate the FITC-BSA and induce a thrombus within the vasculature. In vivo television microscopy was used to quantitate thrombus formation and microvascular diameter changes. The time of platelet thrombus initiation and subsequent time of thrombus growth were studied at wall shear rates of approximately 2000 sec(-1) and 270 sec(-1) in third-order arterioles and venules, respectively. In SHR, times for platelet thrombus initiation and vessel occlusion were significantly less in both arterioles and venules, whereas time for platelet thrombus growth following initiation was significantly prolonged. Greater shear rates in arterioles compared to venules decreased platelet adhesion and subsequently decreased the rate of thrombus formation in both WKY and SHR groups. However, the ratio of WKY to SHR platelet thrombus growth (platelet aggregation) time remained similar (0.83 +/- 0.06 in arterioles and 0.79 +/- 0.06 in venules). These results indicate that there is increased thrombus formation during hypertension and that the platelet adhesion processes may be of greater importance than platelet aggregation in producing this increase.

Increased erythrocyte aggregation in spontaneously hypertensive rats.

Alterations of red blood cell (RBC) aggregation and plasma viscosity are major contributors to the changes in blood rheologic properties that cause an increase in peripheral vascular resistance during the development of hypertension. Although basic research and clinical study have provided considerable understanding of the pathophysiology of hypertension, the objective of this study was to determine whether an increase in RBC aggregability and plasma viscosity precede or accompany the development of high arterial blood pressure. To address this question, RBC aggregation and plasma viscosity were studied in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) at 3 and 12 weeks of age. The plasma concentrations of fibrinogen and fibronectin (FN) were also analyzed in both age groups. RBC aggregability and plasma viscosity were increased in both young and mature SHR compared to age-matched normotensive WKY rats. Mean arterial blood pressure and diastolic pressures were increased in mature hypertensive rats, whereas in young SHR only diastolic pressure was elevated significantly. The concentration of fibrinogen was higher only in the mature hypertensive rats, whereas plasma FN content was greater in both 3- and 12-week-old SHR compared to age-matched WKY. These results show the existence of increased RBC aggregability and plasma hyperviscosity not only during the established phase of hypertension, but also during the early stage of hypertension development, when mean arterial blood pressure is not yet significantly elevated in the genetically hypertensive rat model. These changes may be related to significant increase in the plasma protein FN, which occurs at the same time as the RBC aggregability and plasma viscosity changes. These results may increase attention to changes in the rheologic properties and to the mechanisms involved in these processes in the early stages of hypertension development.

Cadmium-induced arteriolar constriction in skeletal muscle microcirculation.

Cadmium, an environmental pollutant, is known to induce hypertension in animal models, in part via an increase in peripheral vascular resistance. Since prior studies have investigated the vascular effects of cadmium using large, nonresistance arteries, we directly assessed cadmium's action on resistance size arterioles in skeletal muscle using the intact rat cremaster muscle preparation. Cadmium evoked a concentration-dependent constriction of the large arterioles (120 to 50 microns in diameter) but elicited no change in the diameter of smaller arterioles (30 to 15 microns). Blockade of alpha-adrenergic receptors did not diminish the constrictor response of the larger arterioles to cadmium, but bathing the cremaster muscles with a solution containing low calcium attenuated the arteriolar constriction to cadmium. Calcium repletion caused the arterioles to constrict further. These observations provide the first direct evidence that cadmium constricts resistance arterioles in skeletal muscle. The cadmium constriction: (1) is selective for the large arterioles, (2) is not mediated by alpha-adrenergic receptors, and (3) is influenced by the extracellular level of calcium. We conclude that arteriolar constriction in skeletal muscle tissue may play a role in the hypertensive actions of cadmium.

Altered vascular reactivity in arterioles of chronic intermittent hypoxic rats.

Recurrent episodic hypoxia (EH) is a feature of sleep apnea that may be responsible for some chronic cardiovascular sequelae such as systemic hypertension. Chronic EH (8 h/day for 35 days) causes elevation of diurnal resting (unstimulated) mean arterial blood pressure (MAP) in the rat. We used in vivo video microscopy to examine arteriolar reactivity in the cremaster muscle of male Sprague-Dawley rats subjected to 35 days of EH. Cremaster muscles of EH (n = 6) and control (n = 6) rats were exposed to varying doses of norepinephrine (NE) (10(-10) to 10(-5) M), ACh (10(-9) to 10(-5) M), and endothelin-1 (10(-12) to 10(-8) M). In a separate experiment, EH (n = 5) and control (n = 6) rats were given one dose of a nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME; 10(-5) M). We also examined endothelial NOS mRNA from the kidneys of EH-stimulated and control (unstimulated) rats. Telemetry-monitored EH rats showed a 16-mmHg increase in MAP over 35 days, whereas control rats showed no change. The response to NE and endothelin-1 were similar for EH and control rats. ACh vasodilatation of arterioles in EH rats was significantly attenuated compared with that of controls. The degree of vasoconstriction in response to blockade of the nitric oxide system by L-NAME was significantly less (83% of baseline diameter with L-NAME) for arterioles of EH rats compared with that for controls (61% of baseline diameter), implying lower basal resting nitric oxide release in the EH rats. Whole kidney mRNA endothelial NOS levels were not different between groups. These data support the hypothesis that chronic elevation of blood pressure associated with EH involves increased peripheral resistance from decreased basal release or production of nitric oxide after 35 days of EH.

Neuropeptide Y-induced constriction in small resistance vessels of skeletal muscle.

The in vivo responsiveness of small arterioles and venules in the rat cremaster muscle to topical administration of neuropeptide Y was assessed using closed-circuit television microscopy. Male Sprague-Dawley rats were anesthetized with sodium pentobarbital (50 mg/kg) and the cremaster muscle was exposed to increasing bath concentrations of neuropeptide Y (10(-10)-10(-7) M). Neuropeptide Y produced dose-dependent constrictions in first (90 +/- 8 microns), second (50 +/- 6 microns) and third (21 +/- 4 microns) order arterioles. Arteriolar reactivity to the peptide was inversely related to vessel diameters. Venules were relatively unresponsive to neuropeptide Y. Exposure to the alpha-adrenergic receptor antagonist, phentolamine (10(-6) M), failed to modify the arteriolar constrictor responses to neuropeptide Y, while pretreatment with the sympathetic neuronal blocking agent, guanethidine (10(-5) M), produced a small, but significant, reduction in sensitivity. These data suggest that neuropeptide Y causes constriction of arterioles of skeletal muscle, primarily by acting directly on vascular smooth muscle to induce contraction, and not via release of endogenous norepinephrine.

In vivo arteriolar dilation in response to parathyroid hormone fragments.

The in vivo responsiveness of small arterioles to the topical administration of two parathyroid hormone fragments was investigated using television microscopy. Male Sprague-Dawley rats were anesthetized with sodium pentobarbital (50 mg/kg) and second- and third-order arterioles in the cremaster muscle were exposed to increasing concentrations (2 X 10(-5) to 6 X 10(-4) mg/ml) of either hPTH (1-34) or bPTH-(3-34). Second- and third-order arterioles within the cremaster dilated (183% and 281% of control, respectively) following exposure to PTH-(1-34) in bath concentration of 10(-4) mg/ml and above. The dilation associated with PTH administration was abolished in second-order and greatly attenuated for third-order arterioles when the first two amino acid residues of the PTH molecule were removed (PTH (3-34) fragment). Inhibition of endogenous prostaglandins synthesis with mefenamic acid did not attenuate the vasodilator response to PTH. However, exposure to the muscarinic blocking agent atropine (10(-7) g/ml) totally inhibited the dilator response to PTH-(1-34). These data suggest that PTH induces arteriolar dilation by stimulation of muscarinic receptors in the vasculature possibly by causing the release of endogenous acetylcholine.

Met-enkephalin-induced vasoconstriction in the microcirculation of rat striated muscle.

Sprague-Dawley rats were anesthetized with pentobarbital (50 mg/kg) and the arterioles of the cremaster muscle were observed using television microscopy. Methionine (Met)-enkephalin was added to give bath concentrations of 10(-11) to 10(-7) M. Met-enkephalin caused a decrease in the diameter of first-order (127 +/- 7 microns), second-order (57 +/- 7 microns), and third-order (18 +/- 2 microns) arterioles, with the third-order arterioles showing the greatest reactivity. The effects of Met-enkephalin were attenuated by the opiate antagonist, naloxone, by the alpha-adrenergic receptor antagonists, phentolamine and prazosin, and by chemical sympathectomy with 6-hydroxydopamine. These results suggest that Met-enkephalin causes vasoconstriction in the striated muscle microcirculation by stimulating norepinephrine release from local nerve terminals.

Neural release of substance P causes dilation of arterioles in rat striated muscle.

Substance P, which is present in small nerve terminals and proximal to microvessels in rat strated muscle, may have a vasodilator role if released into the microcirculation. Male, Sprague-Dawley rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and the cremaster muscle, with intact blood supply and innervation, was suspended in a bath containing a physiological salt solution. The major autonomic innervation to the cremaster (genito-femoral nerve) was isolated and its cut, distal end was stimulated (3-5 Hz, 2 ms, 10-20 V). Diameters of third order arterioles (14-23 microns) were measured by television microscopy. Stimulation after a 20-min pretreatment with the alpha-adrenergic receptor antagonist phentolamine (2.10(-5) M) unmasked a moderate vasodilation, which was attenuated by treatment with the substance P receptor antagonist [D-Arg1,D-Pro2, D-Trp7,9,Leu11]-substance P (1.10(-6) M). This neurogenic vasodilation was not sensitive to muscarinic receptor blockade by atropine (1.10(-4) M), but was partially blocked by the beta-adrenergic receptor antagonist propranolol (1.10(-5) M). These data suggest that the rat striated muscle microvasculature is innervated with nerves containing substance P, and the release of substance P from the nerve terminals causes arteriolar dilation.

Dilator actions of endothelin in coronary resistance vessels and the abdominal aorta of the guinea pig.

Endothelin has been characterized as a potent constricting factor. The purpose of this study was to investigate possible dilator effects of this peptide and to examine whether dilator responses occur through an endothelium-mediated mechanism in guinea pig coronary resistance vessels and isolated aortic rings. Changes in perfusion pressure after bolus injections of endothelin were measured using a constant-flow modified Langendorff preparation with a transducer between the flow pump and the heart. An immediate fall in perfusion pressure, averaging 6 mmHg, was observed after injection of endothelin (10(-14)-10(-12) moles). This effect was maximal at 1 minute and tended to return toward baseline levels within 4 minutes. In response to endothelin (10(-9) M), isolated aortic rings relaxed 35% after being contracted with prostaglandin F2 alpha (10(-7) M). In both preparations, dilation was converted to constriction after endothelium damage by oxygen radicals or endothelium removal (mechanical rubbing). Dilator responses to endothelin were blocked by pretreatment for 30 minutes with indomethacin (14 microM) in the presence of an intact endothelium in coronary resistance vessels, whereas in the abdominal aorta they were not. We conclude that endothelin has significant dilator properties and that this effect is opposed by its constrictor action at higher doses. In addition, dilator responses to endothelin require an intact endothelium in both coronary vessels and abdominal aorta. Finally, endothelin-induced dilation in coronary resistance vessels appears to occur through a cyclooxygenase product-mediated mechanism.

Vasodilator effect of insulin on the microcirculation of the rat cremaster muscle.

We recently showed that, in conscious rats, acute infusions of insulin (10-15 fold increase in plasma insulin) produced decreases in hindquarter vascular resistance, but only if, changes in sympathetic outflow were prevented with a ganglionic blocker. The aim of the present investigation was to determine if similar effects of insulin could be observed in a preparation that allowed direct visualization of striated muscle (cremaster) microvessels. Initial studies with topical application of insulin showed that third-order arterioles (A3), but not first- or second-order arterioles vasodilated in response to 800 microU/ml and 8 mU/ml of insulin. Systemic (euglycemic) infusion of insulin (6 mU/ml, but not 2 mU/ml) also increased A3 arteriole diameter in animals treated with a ganglionic blocker, but not in control rats. These data show that insulin can have a direct vasodilator effect on striated muscle microvessels if concomitant increases in sympathetic outflow are absent. However, the response was only present with supraphysiological doses of the hormone.

Altered expression and activity of G-proteins, mitogen activated protein kinases, and tyrosine kinases in aging kidney cortex.

BACKGROUND: Renal function declines with age and this may be related to changes in the expression or activity of various signal transduction proteins in the kidney. METHODS: The present study compared the expression and activity of G alpha i(1-3) and G alpha s phosphorylation of mitogen activated protein kinases (MAP-K) (44 and 42 kd) and the activity of tyrosine kinase in renal cortical homogenates of young (4-month-old) and aging (14-month-old) rats. RESULTS: The GTP/(GTP + GDP) binding ratio of G alpha s was significantly decreased in the kidney cortex of aging rats compared to young rats, while the GTP/(GTP + GDP) binding ratio of G alpha i(1-3) increased significantly in kidney cortex of aging rats. Tyrosine kinase activity and phosphorylation of MAP-K (44 and 42 kd) were also reduced in the kidney cortex of aging rats compared to young rats. CONCLUSIONS: These results suggest that diminished phosphorylation of MAP-K and tyrosine kinase activity as well as changes in the binding of GTP/(GTP + GDP) to G alpha i(1-3) and G alpha s may contribute to the age-related decline in renal tubular and vascular function seen in aging animals.

Role of tumor necrosis factor-alpha in small intestinal microcirculation.

The systemic manifestations of sepsis are associated with increased cardiac output, peripheral vasodilatation, and mesenteric vasoconstriction. Our objective was to determine whether tumor necrosis factor (TNF)-alpha regulates small intestinal microcirculatory changes observed during sepsis. An intact loop of terminal ileum of an anesthetized rat was exteriorized into modified Krebs solution and then topically suffused with varying concentrations of TNF-alpha (10(-4) ng/ml to 10(2) ng/ml), norepinephrine (10(-4) M), and sodium nitroprusside (10(-5) M). Videomicroscopy was used to measure arteriolar (A1, A2, A3) and venular (V1, V2) diameter changes in response to topical TNF-alpha. First order vessel diameters did not change in response to TNF-alpha. However, second and third order arterioles dilated maximally by 35 +/- 16 and 52 +/- 12 per cent, respectively, in a dose dependent manner in response to TNF-alpha. Higher order vessels were more sensitive to TNF-alpha than lower order vessels. Norepinephrine (10(-4) M) produced vasoconstriction in all vessels tested (A2 18 +/- 3 per cent, p < 0.05; A3 6 +/- 6 per cent; V2 13 +/- 4 per cent, p < 0.05). Topical TNF-alpha caused dilation in preconstricted vessels as in the nonpreconstricted vessels. TNF-alpha induced vasodilation was prolonged and not reversed by removal of TNF-alpha. These data demonstrate statistically significant dilation in response to TNF-alpha in second and third order arterioles and venules of the small intestine. Persistent vasodilation suggests an induced mechanism of vasodilation in response to TNF-alpha that remains active even after removal of exogenous TNF-alpha. We, therefore, conclude that TNF-alpha causes persistent vasodilatation beyond the period of actual exposure to TNF-alpha in the small intestinal microcirculation. This effect is not altered by the presence of norepinephrine. These data suggest that small intestinal vasoconstriction observed during clinical conditions such as sepsis is unlikely to be mediated by TNF-alpha.

A limited restraint harness for use in long-term infusion of chickens.

A limited restraint harness was developed which permits long-term infusion of chickens under near normal conditions. The harness allows the birds to stand, sit, move forward and backward, and turn 180 degrees in either direction without twisting the cannula.

Lanthanum potentiation of the vascular response to a protein kinase C activator in genetically hypertensive rats.

The effects of lanthanum on the contraction induced by the protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA) were studied in femoral artery rings from stroke-prone, spontaneously hypertensive rats (SHRSP) and normotensive Wistar-Kyoto rats (WKY). When exposed to a calcium-free buffer containing 1 mmol/l EGTA, the femoral artery rings from SHRSP and WKY, pre-contracted with TPA (10(-6) mol/l), relaxed by 52 and 24%, respectively. Treatment of the rings in this calcium-free buffer with 2.6 mmol/l lanthanum significantly potentiated the TPA-induced contractions in vascular rings from WKY (49%) and SHRSP (136%). Potentiation by lanthanum of the TPA-induced contraction in the absence of extracellular calcium suggests that this cation is acting intracellularly to increase protein kinase C activity. The increased vascular responsiveness of SHRSP to lanthanum may reflect an abnormality in protein kinase C activation in vascular smooth muscle of genetically hypertensive rats.

Endothelin-induced vasoconstriction of small resistance vessels in the microcirculation of the rat cremaster muscle.

Recently, a peptide (endothelin) which has been shown to have potent vasoconstrictor properties has been isolated from the vascular endothelium. In the present study, we assessed the responsiveness of small arterioles and venules in the rat cremaster muscle to topical application of endothelin using closed-circuit television microscopy. Exposure to increasing concentrations of endothelin (10(-15)-10(-7) M) produced a dose-dependent constriction in large (90 +/- 8 microns), intermediate (50 +/- 6 microns), and small (21 +/- 4) arterioles. Large (144 +/- 17 microns) and intermediate (79 +/- 18 microns) venules also constricted to the peptide, but the responses were inconsistent and smaller in magnitude. The constriction to endothelin was long lasting and resistant to washout. Arteriolar reactivity to endothelin was similar for all vessel levels with ED50 values ranging from 10(-9) to 10-s;1(0) M. Exposure to the calcium entry blocker, verapamil, attenuated the endothelin-induced constriction in 3A arterioles, suggesting that the constriction in skeletal muscle arterioles is at least partially due to the increased entry of extracellular calcium.