Pressure-induced vasoconstriction of renal microvessels in normotensive and hypertensive rats. Studies in the isolated perfused hydronephrotic kidney

The capacity of small arteries to respond to increased intravascular pressure may be altered in hypertension. In the kidney, hypertension is associated with a compensatory shift in the autoregulatory response to pressure. To directly determine the effects of established hypertension on the renal microvascular response to changes of perfusion pressure, we evaluated pressure-induced vasoconstriction in hydronephrotic kidneys isolated from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Vessel diameters of interlobular arteries (ILAs) and afferent and efferent arterioles were determined by computer-assisted videomicroscopy during alterations in renal arterial pressure (RAP) from 80 to 180 mm Hg. Increased RAP induced a pressure-dependent vasoconstriction in preglomerular vessels (afferent arterioles and ILAs), but not in postglomerular vessels (efferent arterioles). The calcium antagonist nifedipine prevented pressure-induced afferent arteriolar vasoconstriction with a similar half-maximal inhibitory concentration (IC50) (WKY, 63 +/- 27 vs. SHR, 60 +/- 32 nM). The pressure-activation curves for ILAs in SHR and WKY were similar. In contrast, the pressure-activation curve for afferent arterioles in SHR kidneys exhibited a rightward shift, which was observed at every segment of the afferent arteriole (i.e., near ILA, at midportion, and near glomerulus). These findings demonstrate that the ILA and the afferent arteriole both possess the ability to constrict in response to increased pressure, whereas this property is lacking in the efferent arteriole. Hypertension was associated with a compensatory shift in the pressure response of the afferent arteriole, such that higher RAPs were required to elicit vasoconstriction in this vessel.     

Effect of vasodilators on hepatic microcirculation in cirrhosis: a study in the isolated perfused rat liver

We studied the effects of a series of vasodilators on intrahepatic vascular resistance of isolated perfused cirrhotic rat livers in basal conditions and during norepinephrine-induced vasoconstriction. Cirrhosis was induced by repeated intraperitoneal injections of carbon tetrachloride. The vasodilators were a nonselective beta-adrenergic antagonist (propranolol), an alpha 1-adrenergic antagonist (prazosin), a nonselective beta-adrenergic agonist (isoproterenol), an alpha 2-agonist (clonidine), nitrovasodilators (nitroglycerin and nitroprusside), calcium channel blockers (verapamil, diltiazem, nifedipine), papaverine, diazoxide and pentoxifylline. In basal conditions, isoproterenol, nitroglycerin, papaverine, pentoxifylline and nitroprusside demonstrated significant vasodilatory activity. However, the response was weak and isoproterenol was the only drug active in the therapeutic range of concentrations. Propranolol, prazosin, verapamil, diltiazem, nifedipine and diazoxide were ineffective. Prazosin, papaverine and pentoxifylline reduced norepinephrine-induced vasoconstriction, whereas isoproterenol, clonidine and propranolol were ineffective. We conclude that several vasodilators can reduce resistance in the cirrhotic rat liver, but their potency is low and few are effective at therapeutic concentrations. Furthermore, their activity may be blunted when resistance is increased by norepinephrine.     

The hepatic microcirculation in the isolated perfused human liver

In cirrhosis, capillarization of sinusoids could result in impaired exchanges between the hepatocytes and the blood perfusing the liver and contribute to liver failure irrespective of the metabolic capacity of the liver. To characterize anomalies of the hepatic microcirculation, we used the multiple-indicator dilution approach in isolated perfused livers obtained from patients with cirrhosis at the time of transplantation, and from organ donors with normal or near-normal livers or hepatic steatosis. In organ donors, the sinusoidal volume and the permeability of sinusoids to albumin, sucrose, and water were found to be comparable to that of normal dog and rat livers. The sinusoidal volume and the extravascular volume (EVV) accessible to diffusible tracers were larger after hepatic artery than after portal vein injection, probably because of an unshared arterial sinusoidal bed. In cirrhotic livers, two kinds of alterations were found: the appearance of a barrier between the sinusoids and the hepatocytes (capillarization) and intrahepatic shunts. The extravascular space accessible to albumin decreased with increasing severity of cirrhosis, and the diffusion of sucrose in the space of Disse showed a barrier-limited pattern, instead of the normal flow-limited behavior. In cirrhotic livers, a correlation was found between the hepatic extraction of indocyanine green (ICG) and the extravascular space accessible to albumin (r = .84, P < .05), suggesting that the impaired access of this protein-bound dye to the hepatocyte surface contributed to its impaired elimination. Intrahepatic shunts were found between portal and hepatic vein (21% +/- 16% of portal flow), but not between hepatic artery and hepatic veins. We conclude that (1) the behavior of diffusible tracers in human livers with normal liver architecture is comparable to that reported in normal animals; (2) the permeability of sinusoids in cirrhotic livers is abnormal, (3) permeability changes are related to changes in liver function in cirrhosis. (Hepatology 1996 Jan;23(1):24-31)     

Protective action of calcium channel antagonist agents against ventricular fibrillation in the isolated perfused rat heart

This study investigates the effect of calcium channel antagonist agents, verapamil, nifedipine and diltiazem on the ventricular fibrillation threshold before and after left main coronary artery ligation in the isolated perfused rat heart.Verapamil 1.5 × 10^?7m and nifedipine 10^?6m decreased, whereas diltiazem 5 × 10^?6m did not alter the ventricular vulnerability to fibrillation in control non-ligated hearts. During acute regional myocardial ischaemia, verapamil 1.5 × 10^?7m, nifedipine 10^?6m and diltiazem 5 × 10^?6m decreased the vulnerability to ventricular fibrillation. In series paced at 230 beats/min both the d(+) and l(?) optical isomers of verapamil 1.5 × 10^?7m displayed equivalent anti-fibrillatory activity during acute regional myocardial ischaemia. Racemic verapamil 1.5 × 10^?7m displayed greater protection than either isomer alone. The mechanism whereby calcium channel antagonist agents mediate protection against ventricular fibrillation appears exceedingly complex due to the heterogeneity of action of these agents, viz. (1 slowing of heart rate; (2) preservation of high energy phosphate; (3) coronary artery vasodilation; (4) reduction in tissue cyclic AMP content; (5) inhibition of transmembrane calcium influx; and (6) nonspecific effects. Factors which appear to be important in mediating protection against ventricular fibrillation are calcium channel antagonism and a non-specific effect which may represent fast channel inhibition. The relationship between coronary flow distribution to the ischaemic myocardium and the anti-fibrillatory activity of calcium antagonist agents needs to be defined.     

肿瘤坏死因子-α增强内皮素肾血管收缩作用的研究

目的 探讨肿瘤坏死因子-α(TNF-α)及肝素对内皮素-1(ET-1)收缩肾脏血管作用的影响,研究TN F-α在肝肾综合征发病机制中的作用。方法 应用离体灌注肾技术,恒量灌流正常大鼠的离体肾脏,在多导生理记录仪上连续记录肾脏灌注压力的改变,观察经TNF-α,肝素灌流90 min后,在ET-1刺激下,肾脏灌注压的改变。 结果 对照组,应用2 nmol/L ET-1刺激,可使灌注压上升(47±9)mm Hg;TNF-α(1 μg/L)处理组,基线压力无改变,应用2 nmol/L ET-1刺激,可使灌注压上升(97±36)mm Hg,与对照组比较差异有非常显著性,t=3.811,P<0.01;肝素(10 mg/L)处理组,基线压力无改变,应用2 nmol/L ET-1刺激,可使灌注压上升(11±6)mm Hg,与无肝素预灌流组[高出基础灌注压(30 ±6)mm Hg)]比较其灌注压升高幅度明显下降(t=9.41 4,P<0.01)。肾组织HE染色,3组均未见病理改变。 结论 TN F-α可能是通过增强三磷酸肌醇受体表达促进肾血管收缩,在肝肾综合征的发病机制中发挥重要作用。     

Effects of atrial natriuretic peptide on renal function and renin release in the isolated perfused rat kidney

The effects of synthetic atrial natriuretic peptide (ANP) on the renal hemodynamics, glomerular filtration rate (GFR), fluid and electrolyte excretion and renin release were studied in the isolated perfused rat kidney (IPK). When 10(-9) mol of ANP was administered in 75 ml of perfusate, the renal vascular resistance (RVR) was transiently decreased for 3 to 5 min, thereafter increased for 30 min and then tended to return to the control level. ANP increased the GFR (0.55 +/- 0.08 to 0.71 +/- 0.07 ml/min), urine flow (UV) (0.018 +/- 0.002 to 0.194 +/- 0.028 ml/min), absolute Na excretion (UNaV) (1.83 +/- 0.03 to 17.93 +/- 2.71 microEq/min) and absolute K excretion (UKV) (0.67 +/- 0.13 to 2.33 +/- 0.18 microEq/min). The addition of indomethacin or mefenamic acid to the perfusate before the administration of ANP exerted no influence on any of the effects of ANP. Renin release was inhibited by approximately 50% compared to the ANP-free control group. With the administration of ANP, UV and UNaV reached a peak 15-20 min after the GFR reached a peak and remained elevated after the GFR fell below the control level. These findings suggest that 10(-9) mol of ANP causes natriuresis and renin suppression in the IPK, and that the natriuresis is prostaglandin-independent and cannot be explained only by an increase in GFR.     

Influence of calcium on the metabolism of intact parathyroid hormone by isolated perfused rat kidney and liver

The metabolism of synthetic human PTH [PTH-(1-84)] 10(-9) M was studied in isolated rat kidneys and livers, perfused at a calcium concentration of 1 mM or 4 mM. Clearances were measured by an assay specific for intact PTH, and by assays specific for NH2-terminal, mid-molecule, and COOH-terminal immunoreactive PTH (iPTH). Production of PTH fragments was analyzed by HPLC. The kidneys cleared PTH mainly by filtration. The glomerular filtration rate was not lower at 4 mM calcium than at 1 mM calcium, and no significant differences were found between the clearance of PTH at 4 mM and at 1 mM calcium. At 1 mM calcium the kidneys cleared intact PTH without release of detectable fragments. At 4 mM calcium there was significant (P less than 0.05) accumulation of mid-molecule and COOH-terminal iPTH in the perfusate. Both at low and at high calcium the livers cleared NH2-terminal iPTH at the same rate as intact PTH, whereas mid-molecule and COOH-terminal iPTH was cleared significantly (P less than 0.005) slower. In the livers, metabolic clearance of PTH was 60% faster at 4 mM calcium than at 1 mM calcium (P less than 0.001). Assuming that the hepatic metabolism of PTH represents degradation of the biologically active hormone and hormone fragments, rather than activation of the hormone, the present results suggest a homeostatic control of PTH degradation in the liver to enhance inactivation of the hormone at high serum levels of calcium.     

Molecular studies of the calcium antagonist binding site on calcium channels

Currently available calcium antagonists act primarily on L-type calcium channels. Composed of 5 subunits, this ion channel is markedly more complicated than the potassium and sodium channels. Most of the data that have emerged over the past year have concerned the α subunit. The secondary structure of this subunit includes 4 repeating motifs; each motif contains 6 membrane-spanning helices, designated S₁ through S₆. The dihydropyridine (e.g., nifedipine) binding site is most likely situated primarily in motif 4 and the phenylalkylamine (e.g., verapamil) binding site is on motif 4 in an intracellular region. The benzothiazepine (e.g., diltiazem) binding site is also located on the α₁ subunit, but its precise location has not been determined. The transmembrane helices S₅ and S₆ of each motif have been proposed as the “wall” of the actual ion channel.     

Differences in cortical microcirculation in the kidneys of unilaterally congenital hydronephrotic rats

The surgically induced split hydronephrotic kidney has been generally accepted as a valid model for the assessment of renal microcirculation by means of intravital microscopy. Whereas nearly all previous work on this issue has been done with a transillumination technique, we used an epiillumination model that is suitable for investigation of microvascular perfusion in both normal and hydronephrotic kidneys without surgical manipulation of the ureter. By means of the congenital unilaterally hydronephrotic Tauchi rat, microcirculation of the hydronephrotic and that of the nonhydronephrotic kidney were compared. For that purpose both the hydronephrotic and the nonhydronephrotic kidneys of Tauchi rats were exteriorized on a specially designed microscopy stage. After injection of FITC-dextran and rhodamine 6G, microvascular perfusion was assessed in both kidneys. The new model allowed visualization of arterioles, capillaries, and postcapillary venules in both the hydronephrotic and the nonhydronephrotic kidneys. Glomeruli could only be regularly seen in the hydronephrotic kidney, but also in some normal kidneys. Capillary blood cell velocity was significantly higher in the hydronephrotic kidneys (0.67 +/- 0.03 mm/s) compared to the normal kidney (0.32 +/- 0.05 mm/s; P < 0.05), whereas capillary diameters were smaller (4.2 +/- 0.02 microm vs. 5.7 +/- 0.2 microm; P < 0.05). In addition, the hydronephrotic kidney showed a significantly lower density of perfused microvessels compared to the normal controls. Epiillumination intravital microscopy allows assessment of the cortical microcirculation in both the hydronephrotic and the nonhydronephrotic kidneys without surgical induction of hydronephrosis. The hydronephrotic kidney shows significant microcirculatory differences compared to normal kidneys that should be taken into account when using a hydronephrotic model for pharmacological testing.     

Biological effects of aluminum on normal dogs: studies on the isolated perfused bone

Although it is well known that aluminum (Al) plays a role in the development of osteomalacia in patients with chronic renal failure, the mechanisms are not fully understood. Since the osteoblasts are the cells responsible for the formation of osteoid tissue, which is greatly affected in patients with Al-induced osteomalacia, it is possible that Al could affect the number of osteoblasts or interfere with their function. To further characterize this potential mechanism, we performed studies in isolated perfused tibiae from normal and Al-treated dogs. In this system, when PTH is added to the perfusate, cAMP, a major marker of osteoblasts, is released. The dogs were divided into two groups: control, and Al-treated (0.75 mg/kg, iv, 5 days a week for 3 months). Thereafter, the dogs were killed, and the tibiae were perfused in vitro. PTH-(1-34) (3-4 ng/ml) and 3-isobutyl-1-methylxanthine (an inhibitor of phosphodiesterase) were added to the perfusate. Basal cAMP secretion was the same in both groups of dogs. After PTH was added to the perfusate, cAMP increased to a peak of 188.2 +/- 30.6 pmol/min in the normal dogs vs. 113 +/- 8.15 in Al-treated dogs (P less than 0.05). Cumulative cAMP secretion over a 30-min period was 766 +/- 127.9 pmol in the normal dogs vs. 455.6 +/- 38.2 pmol in the experimental animals (P less than 0.05). The histological appearance of bone biopsies taken before and after Al administration are consistent with a suppressive effect of the cation on osteoblast function. In particular, the number of osteoblasts had decreased 8-fold (P less than 0.01) under the influence of Al, and tetracycline-based measurements of mineralization kinetics show that osteoblast-mediated calcification was dysfunctional (P less than 0.01-0.025). On the other hand, the histological features of the post Al treatment biopsies suggest that at some time during its administration, the cation stimulates osteoblastic activity. For example, new (woven) bone formation was present in two dogs, and in another, lamellar bone, deposited under the influence of Al, covered the entire trabecular surface. Moreover, Al-associated osteoid was deposited independent of prior resorptive activity, indicating that the cation promotes bone formation in the absence of prior resorption. In keeping with its trophic effect on matrix deposition, Al also led to extensive marrow fibrosis in five dogs, indicating that Al also stimulates the activity of fibroblasts, cells closely related to osteoblasts.(ABSTRACT TRUNCATED AT 400 WORDS)     

Antihypertensive effects of the putative T-type calcium channel antagonist mibefradil are mediated by the L-type calcium channel Cav1.2

The role of T-type Ca2+ channels for cardiovascular physiology, in particular blood pressure regulation, is controversial. Selective blockade of T-type Ca2+ channels in resistance arteries has been proposed to explain the effect of the antihypertensive drug mibefradil. In the present study, we used a third generation, time- and tissue-specific conditional knockout model of the L-type Ca2+ channel Cav1.2 (Cav1.2SMAKO mice) to genetically dissect the effects of mibefradil on T- and L-type Ca2+ channels. Myogenic tone and phenylephrine-induced contraction in hindlimb perfusion experiments were sensitive to mibefradil in control mice, whereas the drug showed no effect in Cav1.2-deficient animals. Mean arterial blood pressure in awake, freely moving control mice was reduced by 38+/-2.5 mm Hg at a dose of 1.25 mg/kg bodyweight mibefradil, but not changed in Cav1.2SMAKO mice. These results demonstrate that the effect of the putative T-type Ca2+ channel-selective blocker mibefradil on blood pressure and small vessel myogenic tone is mediated by the Cav1.2 L-type Ca2+ channel.     

A quantitative study of fluorescein isothiocyanate-dextran transport in the microcirculation of the isolated perfused rat liver

Hepatic extraction of solutes depends on microvascular angioarchitecture, hemodynamics and solute concentrations. These factors may contribute to the heterogeneity observed in solute transport and uptake in the hepatic lobules. However, predictions of liver extraction based on black-box models require assumptions about these factors and the microvascular transport mechanisms involved. Consequently, the purpose of this study was to investigate solute transport and uptake by hepatocytes. Livers from male Sprague-Dawley rats were perfused at physiological flowrates and portal pressures on the stage of an in vivo microscope using a low-hematocrit Ringer solution. A bolus of fluorescein isothiocyanate-dextrans (17,900, 39,000, 65,600 or 156,900 MW), which are considered inert fluid-phase markers, was injected into the portal vein. Fluorescein isothiocyanate fluorescence, as a measure of solute concentration, was video recorded in periportal or centrivenular regions of the lobules. Spatial and temporal fluorescence data, measured in sinusoids and hepatocytes, were fit to one-dimensional transport models to determine estimates for an intracellular effective diffusion coefficient and for hepatocyte permeability. The calculated effective diffusion coefficients were 2.5 times larger for dextrans less than 66,000 MW, but were not different between the periportal and centrivenular regions. Also, the values did not show the inverse log-log molecular weight dependency for dextrans seen in other microvascular tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

T-type calcium current in electrical activity of cardiomyocytes isolated from rabbit pulmonary vein

INTRODUCTION: Pulmonary veins (PVs) are known to initiate paroxysmal atrial fibrillation. T-type calcium current (I(Ca-T)) has a role in normal and abnormal automaticity of cardiomyocytes. The aim of this study was to evaluate whether I(Ca-T) contributes to PV electrical activity. METHODS AND RESULTS: By whole-cell clamp techniques in rabbit myocytes, I(Ca-T) was identified in 12 (39%) of 31 PV cardiomyocytes with pacemaker activity, 2 (9%) of 23 PV cardiomyocytes without pacemaker activity, and 2 (15%) of 13 atrial myocytes (P < 0.05). Maximum I(Ca-L) and I(Ca-T) densities from PV cardiomyocytes with pacemaker activity were 6.87 +/- 2.17 pA/pF and 1.38 +/- 0.69 pA/pF, respectively. Nickel (40 microM) decreased the spontaneous activity in 5 (36%) of 14 PV cardiomyocytes (3.1 +/- 0.6 Hz vs 2.2 +/- 0.5 Hz, P < 0.05), reduced the amplitudes of delayed after depolarization from 13 +/- 1 mV to 7 +/- 1 mV (n = 4, P < 0.05) and inhibited transient inward currents from 1.2 +/- 0.2 pA/pF to 0.7 +/- 0.1 pA/pF (n = 11, P < 0.01). CONCLUSIONS: We conclude that I(Ca-T) contributes to PV pacemaker activity and triggered activity, which are of functional importance in PV arrhythmogenesis.     

A protective effect of verapamil on the calcium paradox in the isolated perfused rat heart

Experiments were undertaken to examine whether and how verapamil, one of the well known slow-channel calcium-antagonists, protected cardiac muscles against the calcium paradox in isolated perfused rat hearts.Following a 5-min perfusion with calcium-free buffer at pressure of 80 cm H₂O (high flow) and 40 cm H₂O (low flow), severe and mild calcium paradoxical injuries were produced, respectively, by reperfusion with buffer containing 2.5 mm of calcium for 10 min at 80 cm H₂O. When verapamil (1 mg/l) was added to the perfusion medium during both the calcium depletion and repletion periods, a marked protective effect on the mild calcium paradox was observed, evidenced by a higher recovery in the contractility, an about 50% reduction in development of the contracture, and almost complete prevention in release of creatine kinase, an inhibition of tissue calcium accumulation, and much larger tissue stores of high energy phosphates; whereas no protective effect was observed on the severe calcium paradox. In the mild calcium paradox, administration of verapamil up to 2 min after the readmission of calcium was sufficient for protection against the calcium paradox.It was suggested that verapamil could substantially protect heart muscles from injuries associated with the calcium paradox probably due to an inhibition of sudden calcium influx into the cardiac cells in early calcium repletion period.     

Direct renal action of captopril (SQ 14225): Dissociation of natriuretic and vascular actions in isolated perfused rat kidney

Direct effects of captopril on renal function were examined in isolated perfused rat kidneys. Captopril induced a significant increase in urinary volume and urinary sodium excretion (1.8- and 1.7-fold, respectively; both p < 0.005), whereas urinary potassium excretion and renovascular resistance were not significantly changed.Because the perfusion medium lacks angiotensinogen, kininogen and aldosterone, the natriuretic action of captopril in perfused kidneys may not be related to its inhibitory action on angiotensin I converting enzyme or kininase II. Because the natriuresis was not accompanied by changes in renovascular resistance, it is suggested that captopril possesses a direct natriuretic action and that this property may partly explain the mechanism of captopril-induced natriuresis clinically observed.     

Enhancement of the T-type calcium current by hyposmotic shock in isolated guinea-pig ventricular myocytes.

It is known that swelling and shrinkage of cardiac cells can modulate their electrical activity. However, the effects of osmotic manipulation on cardiac T-type calcium current (I(CaT)) has not been previously reported. In this study, we have examined the effects of cell swelling on I(CaT), using the whole cell patch clamp configuration. Isolated guinea-pig ventricular myocytes were swollen by an external hypotonic challenge (0.7 T). We found that I(CaT)is enhanced during a hypotonic shock. This current has been determined to be the T type calcium current since it is rapidly activated and inactivated, its threshold was at negative potentials and was blocked by 40 microm Ni2+. Disruption of microfilaments by cytochalasin D and of microtubules by colchicine prevented the activation of I(CaT)during cell swelling. Taxol had no effect. These results indicate that I(CaT)is increased during cell swelling and this effect needs an intact cytoskeleton.     

Effects of angiotensin I converting enzyme inhibitors on the renal excretory function, hemodynamics and renin release in isolated perfused rat kidney

Direct renal effects of angiotensin I converting enzyme inhibitors (CEIs), captopril, SA446 and MK421, were examined in isolated rat kidneys perfused with a renin-substrate-free medium. Among three CEIs, only captopril induced a significant natriuresis, whereas SA446 and MK421 did not. UKV, renal vascular resistance and creatinine clearance were not affected by any of these CEIs. Renin release from perfused rat kidneys were not influenced by CEIs under the present experimental conditions. These results suggest that among three different types of CEIs, only captopril possesses natriuretic action in the isolated perfused rat kidney and that this action may be independent of its inhibitory action on angiotensin converting enzyme. It is also suggested that these three CEIs themselves do not have a direct effect on the renal vascular bed.