Jack W. Strandhoy

Transendothelial permeability of chlorpyrifos in RBE4 monolayers is modulated by astrocyte-conditioned medium

The immortalized rat brain endothelium 4 (RBE4) cell line preserves many features of the in vivo brain endothelium. It has been used as an in vitro model of the blood-brain barrier (BBB). Astrocyte-endothelial cell interactions are crucial for maintenance of BBB characteristics. The present study investigated morphological and permeability properties of the RBE4 cell line. Immunohistochemical studies showed positive staining in RBE4 cells for E-cadherin, a Ca(2+)-dependent cell-cell adhesion molecule. Western blot immunoassay showed that RBE4 cells consistently express E-cadherin and that its expression significantly increased (P<0.001) in the presence of astrocyte-conditioned medium (ACM). The transendothelial permeability of chlorpyrifos, an organophosphorus insecticide, was significantly decreased (P<0.001) when the RBE4 cells were grown in ACM compared with control medium. Additional studies were carried out to determine whether chlorpyrifos is a substrate for the multidrug resistance protein, P-glycoprotein (P-gp). No significant change in chlorpyrifos transendothelial permeability was noted in the presence of verapamil, a P-gp blocker. Thus, in this system, chlorpyrifos is not a substrate for P-gp. This work demonstrates that with additional refinements the RBE4 monolayers might serve as a useful in vitro model for the study of BBB permeability and modulation by astrocyte-derived soluble factors.

Renal alpha 1- and alpha 2-adrenoceptor mediated vasoconstriction in dogs: comparison of phenylephrine, clonidine, and guanabenz.

The alpha 2-agonist, guanabenz, increases Na and water excretion with little change in renal blood flow, while clonidine has been reported to cause antinatriuresis proportional to renal vasoconstriction. We hypothesized that clonidine-mediated renal vasoconstriction involves alpha 1-receptors. Dose-response curves were constructed correlating decreases in renal blood flow with doses of phenylephrine, clonidine, and guanabenz injected as boluses into renal arteries of anesthetized dogs. Changes in renal blood flow before and after prazosin, yohimbine or verapamil were recorded. Guanabenz was a 10-fold weaker vasoconstrictor than clonidine, but was still effective when alpha 1-receptors were blocked. Tenfold differences were found between phenylephrine, clonidine, and guanabenz for dependence on alpha 1-receptors. Denervation did not significantly shift the curves. Verapamil markedly attenuated only clonidine and guanabenz, which supported their dependence on calcium channels for vascular smooth muscle contraction. Thus, postsynaptic alpha 2-adrenoceptors can contribute to renal vasoconstriction in the dog but the receptors are either less numerous on the vasculature or are less efficiently coupled to contractile elements than are alpha 1-receptors. Clonidine constricts the renal vasculature through both adrenoceptor subtypes.

The effects of prostaglandins E2 and F2α on synaptosomal accumulation and release of 3H-norepinephrine

Prostaglandins (PG) of both the E and F series may serve as modulators of norepinephrine (NE) release from peripheral sympathetic neurons. We have studied the effects of PGE2 and PGF2alpha on the accumulation and release of 3H-NE in the CNS using synaptosomes isolated from rat hypothalami. The release of 3H-NE from synaptosomes superfused with Krebs-Ringer bicarbonate buffer was multiphasic with an initial fast release phase followed by a slower release. Raising KCl concentration of the superfusion medium to 56mM during the slow release phase is known to stimulate 3H-NE release. PGE2 (1 X 10(-6)M) attenuated 3H-NE release during the fast phase and reduced the amount of 3H-NE released due to KCl stimulation. At lower concentrations of PGE2 there was no change in the release profile. PGF2alpha was without effect on 3H-NE release at all concentrations tested. The accumulation of 3H-NE was significantly diminished by PGE2 at a concentration of 1 X 10(-6)M, while a lower concentration (1 X 10(-7)M) was ineffective. PGF2alpha had no effect on 3H-NE accumulation at all concentrations investigated.

Role of alpha-2 receptors in the regulation of renal function.

Postsynaptic binding sites for α1 and α2 adrenoceptor ligands are found in abundance in the renal cortex of several species, with reports of 2–3 times as many α1 as α2 sites. These α adrenoceptor subtypes can potentially influence salt and water excretion through both vascular and tubular effects. Renal vascular resistance in dogs is increased by both α adrenoceptor subtype agonists but α1 agonists are more potent. In rats, α2 agonists have almost no effect on the renal circulation whereas α1 agonists are capable of intense renal vasoconstriction. The mechanisms by which α2 agonists increase glomerular filtration rate are not yet clear and may involve the secondary release of hormones affecting glomerular dynamics and permeability. Thus, an abundance of α2 adrenoceptor binding sites in whole cortical homogenate of rat kidneys with little demonstrable vascular effect of α2 agonist suggests that the preponderance of these receptors lies instead on the tubular epithelium. Alpha-1 adrenoceptors are probably responsible for the increased Na reabsorption from the proximal tubule and the anti-natriuresis following low level renal nerve stimulation. In contrast, an α1 agonist such as guanaben/produces a diuresis by reducing the release of vasopressin and by antagonizing its hydrosmotic effect on the nephron and a modest natriuresis by decreasing medullary interstitial osmolality and reducing passive Na reabsorption

Regulation of in vitro renin secretion by ANG II feedback manipulation in vivo in the ovine fetus

The renin-angiotensin system is critically important to fetal cardiovascular function and organ development. The feedback regulation of renin secretion by ANG II develops early in gestation yet does not linearly progress from fetal life to adulthood. Renin secretion is elevated in late gestation compared with earlier or postnatal time periods, which suggests that some component of the negative feedback regulation of renin secretion is less sensitive in late gestation. We examined in fetal sheep the age-related consequence of chronic in vivo manipulation of ANG II on renal renin secretion measured in vitro. Immature (101-103 days of gestation) and mature (130-133 days of gestation) fetuses were treated for 72 h with enalaprilat, ANG II or vehicle. Content and basal and isoproterenol-stimulated secretion of prorenin (PR) and active renin (AR) from fetal kidney cortical slices were determined. Enalaprilat pretreatment in vivo increased renal renin content and basal and stimulated secretion of PR and AR in vitro even in immature animals. Immunohistochemical localization showed that enalaprilat treatment caused an age-related recruitment of renin-containing juxtaglomerular cells. Conversely, ANG II pretreatment decreased basal and stimulated PR and AR secretion from immature fetal kidneys, but only inhibited PR secretion from mature kidneys. It also caused an age-related decrease in the percentage of renin-containing juxtaglomerular cells. These results suggest that ANG II feedback modulates not only the synthesis and content of renin, but the sensitivity of the coupling between stimulus and secretion. A critical observation of our study is that the higher renal tissue concentrations of prorenin and active renin in late gestation may be a consequence of reduced sensitivity to ANG II feedback; this is consistent with the increased plasma concentrations of renin found in near-term mammals.The renin-angiotensin system is critically important to fetal cardiovascular function and organ development. The feedback regulation of renin secretion by ANG II develops early in gestation yet does not linearly progress from fetal life to adulthood. Renin secretion is elevated in late gestation compared with earlier or postnatal time periods, which suggests that some component of the negative feedback regulation of renin secretion is less sensitive in late gestation. We examined in fetal sheep the age-related consequence of chronic in vivo manipulation of ANG II on renal renin secretion measured in vitro. Immature (101-103 days of gestation) and mature (130-133 days of gestation) fetuses were treated for 72 h with enalaprilat, ANG II or vehicle. Content and basal and isoproterenol-stimulated secretion of prorenin (PR) and active renin (AR) from fetal kidney cortical slices were determined. Enalaprilat pretreatment in vivo increased renal renin content and basal and stimulated secretion of PR and AR in vitro even in immature animals. Immunohistochemical localization showed that enalaprilat treatment caused an age-related recruitment of renin-containing juxtaglomerular cells. Conversely, ANG II pretreatment decreased basal and stimulated PR and AR secretion from immature fetal kidneys, but only inhibited PR secretion from mature kidneys. It also caused an age-related decrease in the percentage of renin-containing juxtaglomerular cells. These results suggest that ANG II feedback modulates not only the synthesis and content of renin, but the sensitivity of the coupling between stimulus and secretion. A critical observation of our study is that the higher renal tissue concentrations of prorenin and active renin in late gestation may be a consequence of reduced sensitivity to ANG II feedback; this is consistent with the increased plasma concentrations of renin found in near-term mammals.

Vasorelaxant effect of C16-PAF and C18-PAF on renal blood flow and systemic blood pressure in the anesthetized rat.

The renal vasoactive and systemic hypotensive effects of platelet activating factor (C16:0-PAF and C18:1-PAF) were examined in anesthetized male Wistar rats. Bolus injections of C16-PAF (0.5-25 ng/kg) and C18-PAF (2.5-200 ng/kg) into the arterial circulation of the kidney produced increases in renal blood flow (6-15%) before causing dose-dependent systemic hypotension (2-64 mmHg). The dose-response curves for renal blood flow and systemic blood pressure generated by intrarenal C18-PAF administration were approximately 7 fold to the right of the dose-response curves generated by C16-DPAF. Intrarenal injections of vehicle or the biologically inactive enantiomer C16-DPAF (25-200 ng/kg) did not affect renal blood flow or systemic blood pressure. These results suggest that C16:0-PAF is a more potent renal vasodilator and hypotensive lipid than C18:1-PAF.

Antagonism of the hydrosmotic effect of vasopressin by the antihypertensive, guanabenz

Abstract The antihypertensive drugs guanabenz and clonidine have been shown to produce water diuresis. Decreased release of vasopressin (ADH) and reduced tubular Na transport may contribute to the diuretic effect, but a cellular antagonism of ADH has not been excluded. We examined in toad bladders the ability of guanabenz and clonidine to antagonize the increase in osmotic water flow (Jv) which was stimulated by ADH. Guanabenz (1 × 10 −8 to 10 −6 M) decreased ADH-stimulated Jv but not basal Jv. Its primary metabolite had no effect. Clonidine inhibited ADH-stimulated Jv less than guanabenz. The effect of guanabenz was abolished by phenoxybenzamine and yohimbine but not by naproxen or prazosin. Phenylephrine, an α 1 -agonist, decreased ADH-stimulated Jv onlyt or above 1 × 10 −5 M. Guanabenz was about 10-fold less potent an inhibitor of 14 C-urea flux than of osmotic water flow and did not affect short circuit current. These studies suggest that postsynaptic α 2 -adrenoceptors may modulate the action of ADH to increase water permeability of the toad bladder.

Tetrodotoxin protects against acute ischemic renal failure in the rat

Tetrodotoxin has been reported to cause prolonged systemic hypotension without resultant ischemic damage. We tested its ability to protect the kidney during 60 minutes of warm ischemia in uninephrectomized rats. Protection was observed when tetrodotoxin was given intravenously at two microgram./kg. and four microgram./kg. as assessed by serial plasma blood urea nitrogen and creatinine measurements over two weeks. Tetrodotoxin was protective when given immediately before or immediately after the ischemic period. The renal protection of tetrodotoxin was not due to its effects on renal nerves as renal denervation did not protect the kidney from the ischemic damage. The renal protective effects of four microgram. tetrodotoxin/kg. were similar to those of four mg. captopril/kg. but the combination of the two was paradoxically without effect. We tested whether tetrodotoxin and captopril chemically antagonized each other, but in the presence of tetrodotoxin, captopril was still a potent inhibitor of the conversion of angiotensin I to angiotensin II. These results indicate that tetrodotoxin could be useful in elucidating the sequence of events associated with ischemic-reperfusion renal injury and in identifying ways of preserving renal function during renal surgery.

Comparison of the renal effects of indoramin and prazosin.

Indoramin and prazosin are both potent and selective α1 adrenoceptor antagonists. Indoramin has been reported to decrease baroreceptor sensitivity, whereas prazosin has been reported to increase it. The current studies examined the relative potencies of the two drugs in inhibiting the renal vascular α1 adrenoceptor and also the renal and cardiovascular effects of these agents. The drugs were infused intravenously at rates in which they equally antagonized α1-receptors. Indoramin decreased blood pressure by as much as 29 mm Hg with the doses tested and also significantly (p < 0.05) reduced heart rate. Sodium, potassium, and water excretion, and glomerular filtration rate (GFR) were unchanged. Prazosin tended to reduce blood pressure, but the 11 mm Hg fall was not statistically significant. Heart rate increased significantly (p < 0.05). Sodium, potassium, and water excretion decreased, with a return toward control values of urine flow but not sodium excretion with higher doses. The GFR was unchanged. The results of this study indicate that neural and endocrine factors modify direct drug effects and that indoramin and prazosin administered acutely differ in the recruitment of these reflexes.

Clearance and micropuncture studies of the diuretic xipamide in dogs

Effects of the diuretic xipamide were examined by clearance and micropuncture techniques, and compared to clorothiazide. Xipamide decreased GFR and phosphate excretion. Reabsorption by superficial proximal tubules was unchanged. Since xipamide reduced single nephron filtration rate and GFR, a small effect on the proximal tubule may have been masked. Chlorothiazide decreased GFR but additionally decreased reabsorption by the proximal tubule. Xipamide was more chloruretic than natiuretic and probably inhibits reabsorption in the distal tubule.