The stimulation of renin secretion by diazoxide in the isolated rat kidney.

The intrarenal effect of diazoxide on renin secretion was examined in the isolated perfused rat kidney. Mean renin secretion measured at 2 min intervals during the continuous infusion of diazoxide for 6 min was consistently higher than the corresponding control values, although this was significantly different at the end of the infusion only. Since renal perfusion pressure during diazoxide infusion and control studies decreased to a similar extent, it is suggested that diazoxide stimulates renin secretion by a direct effect on the juxtaglomerular cell.     

Intrarenal stimulation of renin secretion by frusemide in the isolated kidney of the rat.

1. Intrarenal infusion of frusemide markedly stimulated renin secretion in the isolated perfused kidney of the rat. 2. Renin vales increased from 24+/-6 to 195+/-34 units of secretion rate (renin concentration (nmol angiotensin I/h per litre) X flow rate (ml/min)) following administration of frusemide for 8 min, compared with corresponding control values of 13 +/- 2 (P greater than 0.05) and 47 +/-18 (P less than 0.001). This stimulatory effect was also observed when urine flow was interrupted by ligation of the ureters. 3. The changes in renal perfusion pressure and perfusate flow rate were not significantly differnt from the control values. 4. These findings indicate the existence of an intrarenal site of action for frusemide on renin secretion. 5. Since frusemide did dot appear to alter perfusion pressure or flow rate, and was effective when urine flow was abolished, a dominant role for a vascular or macula densa receptor mechnaism seems unlikely. A direct effect of frusemide on the renin secreting cell is therefore suggested.     

Inhibition of renin release by alpha-adrenoceptor stimulation in the isolated perfused rat kidney

The effect of the specific alpha 2-adrenoceptor agonist BHT 933 on stimulated renin release was investigated in the isolated perfused rat kidney preparation. Renin release was stimulated with N-ethylcarboxamide adenosine (NECA) (3 microM) a specific A2-adenosine receptor agonist. alpha 2-Adrenoceptor stimulation with BHT 933 (1 microM) attenuated the stimulation of renin release by NECA. Yohimbine (300 nM) or prazosin (28 nM) at alpha 2- and alpha 1-adrenoceptor specific concentrations respectively, blocked this inhibition of renin release by BHT 933. In all groups studied there was no significant effect of these experimental treatments on renal hemodynamics or electrolyte excretion. The ability of yohimbine or prazosin, at alpha 2- and alpha 1-adrenoceptor specific concentrations respectively, to antagonize the effects of BHT 933 suggests a lack of agonist specificity for these receptor effect as previously suggested for the mesenteric artery.     

Failure of alpha-adrenergic stimulation by phenylephrine to enhance renin secretion in the isolated rat kidney.

The intrarenal effect of the alpha-receptor agonist phenylephrine on renin secretion was examined in the isolated rat kidney. Infusion of phenylephrine in non-vasoconstrictor doses resulted in secretion rates which were not significantly different from control values. Similarly no change in renin secretion was found when phenylephrine was infused at a dose that clearly caused renal vasoconstriction and increased vascular resistance. These results do not support the hypothesis of a role for the alpha-receptor in the stimulation of renin secretion by adrenergic activity.     

Alpha 2-adrenoceptor stimulation affects total glucose utilization in isolated islets of Langerhans

Glucose utilization in isolated islets of Langerhans of the rat was determined by measuring the conversion of [5-3H]glucose (10 mM) to 3H2O. The alpha 2-adrenoceptor agonists clonidine, epinephrine, and norepinephrine in the presence of the alpha 1-adrenoceptor antagonist prazosin and the beta-adrenoceptor antagonist propranolol inhibited glucose utilization by as much as 50%. Yohimbine, an alpha 2-adrenoceptor antagonist, reversed the reduction in glucose utilization evoked by alpha 2 receptor agonists. The cholinomimetics carbachol and muscarine, and 8-bromo-cyclic GMP, but not other cyclic nucleotides, reversed the clonidine-induced suppression of glucose utilization. 3-Isobutyl-1-methylxanthine potentiated the stimulation of glucose utilization by carbachol with clonidine. In contrast, the beta-adrenoceptor agonist isoproterenol did not affect glucose utilization. Forskolin, which activates adenylate cyclase, reduced glucose utilization and did not affect the inhibitory response to clonidine. The ester phorbol 12,13-dibutyrate induced a latent reversal of the effects of clonidine. Insulin release paralleled changes in glucose utilization with alpha 2-adrenoceptor agonists. Carbachol and 8-bromo-cyclic GMP antagonized the alpha 2-adrenoceptor-induced inhibition of insulin release. During sustained insulin release (60 min), 8-bromo-cyclic AMP became a more potent modulator of secretion than 8-bromo-cyclic GMP in the presence of clonidine, although glucose utilization was not enhanced by 8-bromo-cyclic AMP.     

The postnatal development of adrenoceptor responses to agonists and electrical stimulation in rat isolated atria.

1. Isolated right and left atria from rats of ages ranging from newborn to adult were used to measure chronotropic and inotropic responses to noradrenaline, isoprenaline, tyramine, and electrical stimulation of intramural nerves. 2. Right atria from newborn animals showed increases in rate with noradrenaline, isoprenaline, and tyramine which did not differ significantly from those of atria from adults. The ED50 values for the chronotropic actions of noradrenaline and isoprenaline were not significantly different at any age from the values in adult preparations. 3. Paced left atria from newborn rats showed well developed positive inotropic responses to noradrenaline and isoprenaline. Newborn left atria (and those from 1 and 2 week old animals) were supersensitive to noradrenaline but not to isoprenaline. 4. Left atria from newborn animals showed very small inotropic responses to both tyramine and field stimulation of intramural nerves. These responses developed progressively with age over the first three weeks of life. The results are discussed with respect to the development of cardiac beta-adrenoceptors and of cardiac sympathetic innervation.     

Effects of GABA on noradrenaline release and vasoconstriction induced by renal nerve stimulation in isolated perfused rat kidney.

We examined effects of gamma-aminobutyric acid (GABA) on vasoconstriction and noradrenaline (NA) release induced by electrical renal nerve stimulation (RNS) in the isolated pump-perfused rat kidney. RNS (1 and 2 Hz for 2.5 min each, 0.5-ms duration, supramaximal voltage) increased renal perfusion pressure (PP) and renal NA efflux. GABA (3, 10 and 100 microM) attenuated the RNS-induced increases in PP by 10-40% (P<0.01) and NA efflux by 10-30% (P<0.01). GABA did not affect exogenous NA (40 and 60 nM)-induced increases in PP. The selective GABA(B) agonist baclofen (3, 10 and 100 microM) also attenuated the RNS-induced increases in PP and NA efflux, whereas the RNS-induced responses were relatively resistant to the selective GABA(A) agonist muscimol (3, 10 and 100 microM). The selective GABA(B) antagonist 2-hydroxysaclofen (50 microM), but not the selective GABA(A) antagonist bicuculline (50 microM), abolished the inhibitory effects of GABA (10 microM) on the RNS-induced responses. The selective alpha2-adrenoceptor antagonist rauwolscine (10 nM) enhanced the RNS-induced responses. GABA (3, 10 and 100 microM) potently attenuated the RNS-induced increases in PP by 40-60% (P<0.01) and NA efflux by 20-50% (P<0.01) in the presence of rauwolscine. Prazosin (10 and 30 nM) suppressed the RNS-induced increases in PP by about 70-80%. Neither rauwolscine (10 nM) nor GABA (10 microM) suppressed the residual prazosin-resistant PP response. These results suggest that GABA suppresses sympathetic neurotransmitter release via presynaptic GABA(B) receptors, and thereby attenuates adrenergically induced vasoconstriction in the rat kidney.     

Alpha2-adrenoceptor antagonists evoke endothelium-dependent and -independent relaxations in the isolated rat aorta.

This study was designed to determine whether the alpha2-adrenoceptor antagonists idazoxan, yohimbine, and rauwolscine cause endothelium-dependent and -independent responses in the rat aorta. Rings of rat aorta, with and without endothelium, were suspended for the measurement of isometric force in modified Krebs-Ringer bicarbonate solution (37 degrees C; aerated with 95% O2 and 5% CO2). The alpha2-adrenoceptor antagonists, in the concentration range of 10(-8)-10(-6) M, relaxed phenylephrine-contracted rings with, but not those without endothelium. alpha2-Adrenoceptor antagonists (3 x 10(-6) M for 1 min) increased the accumulation of cyclic guanosine monophosphate (cGMP) about twofold in the aortas with endothelium. The relaxation and the increased cGMP induced by alpha2-antagonists were attenuated by methylene blue (10(-6) M) and N(G)-nitro-L-arginine (L-NA, 3 x 10(-5) M), whereas propranolol (10(-6) M) did not affect the relaxation. In concentrations >10(-6) M, alpha2-adrenoceptor antagonists relaxed the rat aorta without endothelium. The endothelium-independent relaxation by alpha2-adrenoceptor antagonists was abolished by increased external K+ and reduced significantly by tetraethylammonium (TEA, 10(-2) M, a Ca2+-dependent K+ channel blocker), but not inhibited by glibenclamide (10(-5) M, an ATP-sensitive K+ channel blocker). In the rabbit aorta, only endothelium-independent relaxations were observed with alpha2-adrenoceptor antagonists in the concentration range of 10(-8)-10(-5) M, and these relaxations were not antagonized by TEA. These results suggest that alpha2-adrenoceptor antagonists relax the rat aorta through endothelium-dependent mechanism at lower concentrations and endothelium-independent mechanisms at higher concentrations. The endothelium-dependent relaxations are likely to be mediated by the endothelium-derived relaxing factor (EDRF)/NO pathway because they are associated with the accumulation of cGMP, whereas the endothelium-independent relaxations may be caused by the opening of potassium channels in the vascular smooth muscle.     

Alpha 2U-globulin: measurement in rat kidney following administration of 2,2,4-trimethylpentane

Hyaline droplet formation was stimulated markedly in the kidneys of post-puberty male rats 24-48 h after a single oral dose of 12/24 mmol/kg 2,2,4-trimethylpentane [TMP]. Renal hyaline droplet formation could not be detected in female rats or in pre-puberty male rats following similar doses of TMP. A dose-dependent increase in the renal concentration of the androgen-dependent low molecular weight protein, alpha 2U-globulin was observed in post-puberty male rats 24 h after a single oral dose of TMP, over the range 0.3-12.0 mmol/kg. After administration of a single dose of 12 mmol/kg TMP to male rats, the renal concentration of alpha 2U-globulin rose steadily up to a peak after 48 h and then returned slowly to near normal after 7 days. Renal alpha 2U-globulin could not be detected in female rats and in pre-puberty male rats. An immunocytochemical assay was developed to examine the distribution of alpha 2U-globulin within the kidney. alpha 2U-Globulin was localised primarily in the S2 segment of renal proximal tubules in untreated male rats. Rats which received a single dose of 12 mmol TMP/kg showed not only a greater staining intensity, due to the presence of a higher concentration of alpha 2U-globulin, but also staining in adjacent segments of the renal cortex. Several urinary biochemical indicators of nephrotoxicity were measured daily in male rats for up to 72 h following a single dose of 12 mmol TMP/kg. Renal proximal tubular function was unimpaired by TMP treatment. On the basis of studies in untreated and TMP-treated rats, a strong association has been found between the presence of renal hyaline droplets and the occurrence of renal alpha 2U-globulin. The findings in the present study provide an explanation for the occurrence of renal hyaline droplets only in adult male rats, but do not, as yet, establish the toxicological significance of increases in renal hyaline droplet formation.     

In vitro measurement of endogenous norepinephrine release from small blood vessels with short stimulation trains.

We have previously developed a low-volume perfusion-superfusion system for studying neurotransmitter release in small blood vessels. We now extend this technique to allow for the measurement of neurotransmitter release with short stimulation trains in order to examine modulating factors such as activation of prejunctional receptors or the effects of altered external calcium. Segments of rat tail artery were perfused in the presence of deoxycorticosterone and cocaine (10(-5) M). For short trains, nerves were activated with five sequential trains, each 4 sec in length (total of 160 pulses) at 8 Hz. For long trains, nerves were activated continuously for 3 min for a total of 1440 pulses. Norepinephrine in the perfusate and tissue norepinephrine were quantitated with high-performance liquid chromatography (HPLC) and electrochemical detection. Fractional norepinephrine release was significantly greater with short as compared to long trains when extracellular calcium was maintained at 1.6 mM. When nerves were stimulated with short trains, fractional norepinephrine release was very sensitive to altered extracellular calcium, with a significant decline when extracellular calcium was reduced to 1 mM and a significant increase when extracellular calcium was increased to 5 mM. In contrast, with long-stimulation trains increases or decreases in calcium had no effect on fractional norepinephrine release. This unique method facilitates the study of norepinephrine release with short trains or low-frequency activation and will also make it easier to study presynaptic receptor function as well as interactions between extracellular calcium and presynaptic effectiveness.     

Selective stimulation of glucagon secretion by beta 2-adrenoceptors in isolated islets of Langerhans of the rat.

1. In rat isolated islets of Langerhans the selective beta 2-adrenoceptor agonist, clenbuterol (1 to 20 microM), significantly increased the level of adenosine 3':5'-cyclic monophosphate (cyclic AMP) within 2 min of incubation. 2. The cyclic AMP response to clenbuterol was inhibited in the presence of the selective beta 2 adrenoceptor antagonist, ICI 118551 (0.1 or 10 microM) but remained unchanged when the beta 1-antagonist, atenolol (0.1 microM) was administered. 3. Despite causing an elevation in cyclic AMP, clenbuterol (up to 20 microM) failed to influence insulin secretion at any glucose concentration tested, even in the presence of a phosphodiesterase inhibitor. 4. By contrast, clenbuterol elicited a dose-dependent rise in the rate of glucagon secretion; the maximal agonist-induced increase in secretion was two fold, a response equivalent to that observed with 20 mM L-arginine. 5. ICI 118551 significantly inhibited the rise in glucagon secretion induced by clenbuterol (up to 20 microM). 6. The results indicate that the rat islet A cell population is equipped with functional beta 2-adrenoceptors which influence glucagon secretion via the second messenger cyclic AMP, but that the B cells are deficient in functional beta-receptors.     

The effect of alpha 2-adrenoceptor agonists on the acid secretory responses of rat isolated gastric mucosa to electrical field stimulation.

The effects of clonidine, UK-14,304, noradrenaline, para-aminoclonidine and phenylephrine were examined on the acid secretory response of the rat isolated gastric mucosa preparation to electrical field stimulation. Clonidine, UK-14,304, noradrenaline and para-aminoclonidine but not phenylephrine (10 microM) reduced the response of the gastric mucosa stimulated at 2.5 Hz; gastric mucosae stimulated at higher frequencies were insensitive to the action of these alpha 2-adrenoceptor agonists. The inhibitory effect of the selective alpha 2-adrenoceptor agonist UK-14,304 was antagonized by idazoxan but not by prazosin. These findings indicate that clonidine and other alpha 2-adrenoceptor agents inhibit the acid secretory response of the rat gastric mucosa to electrical field stimulation by an action at alpha 2-adrenoceptors, which are probably located on cholinergic nerve terminals.     

Isoproterenol and nucleotide induced stimulation of Ca2+ uptake by microsomal fractions from kidney and isolated glomeruli.

Renal cortical microsomal vesicles possess an ATP-dependent Ca²⁺ uptake system which is two to three times more active in accumulating Ca²⁺ than are the microsomes prepared from the outer medulla or papilla of the cat kidney. The microsomal Ca²⁺ uptake system was unaffected by sodium azide, and electron microscopy confirmed the absence of intact mitochondria. Ca²⁺ accumulating activity was significantly increased by 13 per cent in cortical microsomes prepared from kidneys which had been perfused with isoproterenol (2 × 10^?7 M), whereas medullary or papillary microsomal Ca²⁺ accumulation was unaffected. Perfusate containing a higher isoproterenol concentration (2 × 10^?6 M) stimulated cortical as well as papillary microsomal Ca²⁺ uptake by 13 and 31 per cent respectively, but had no effect on medullary microsomal Ca²⁺ accumulation. A lower isoproterenol concentration (2 × 10^?8 M) did not change the Ca²⁺ uptake activity of microsomes isolated from either region of the cat kidney. The isoproterenol concentrations (2 × 10^?7 and 2 × 10^?6 M) which activated Ca²⁺ uptake in microsomes produced graded increases in renin secretion, whereas 2 × 10^?8 M isoproterenol was relatively inactive in eliciting renin secretion. Renal cortical tissue exposed to cyclic AMP (cAMP) and 5'-AMP during subcellular fractionation showed significant increases in microsomal Ca²⁺ uptake. However, microsomes exposed to cAMP or 5'-AMP in the Ca²⁺ uptake medium were not stimulated. Isoproterenol also activated Ca²⁺ uptake by microsomes prepared from isolated glomeruli, and this stimulation was blocked by propranolol. We conclude that the cat renal cortex possesses specific receptors for isoproterenol which activate Ca²⁺ transport through a nucleotide mediated mechanism.     

Contractile responses of isolated guinea-pig vas deferens to trains of electrical stimuli and influence of prostaglandin E2

The contractile responses of isolated guinea-pig vas deferens to long trains of 300 stimuli (0.3 ms, 90 V) applied at a frequency of 8 Hz or 20 Hz were diphasic contractions comprising a non-adrenergic, probably ATP-mediated component (phase I) and an adrenergic component (phase II). Stimulation with short trains of 10 stimuli (1 ms, 90 V) at a frequency of 10 Hz elicited monophasic responses. The contractions evoked by either long or short trains of stimuli were due to excitation of sympathetic nerve terminals without, however, being purely adrenergic ones. Prostaglandin E2 (PGE2) in increasing concentrations of 1 nmol/l to 100 nmol/l reduced phase I of the responses to long trains of stimuli at a frequency of 8 Hz, as well as the monophasic responses to short trains of stimuli. Both phases of the contractions evoked by long trains of stimuli at a frequency of 20 Hz were reduced by the lowest and potentiated by the highest concentrations of PGE2. The high concentration of PGE2 also potentiated phase II of the responses to long trains of stimuli at a frequency of 8 Hz and the diphasic contractions, resulting from the simultaneous application of ATP and noradrenaline. The results suggest that in guinea-pig vas deferens, PGE2 in low concentrations presynaptically inhibits the non-adrenergic components of the contractile responses to electrical stimulation of the sympathetic nerve terminals, while in high concentrations postsynaptically potentiates mainly the adrenergic components of these responses.     

Generation of 8-epi-prostaglandin F(2alpha) in isolated rat kidney glomeruli by a radical-independent mechanism.

Isoprostanes comprise a group of free radical-catalyzed products of arachidonic acid. However, there is recent evidence pointing towards an enzyme-dependent formation of isoprostanes. With the use of isolated rat glomeruli we addressed the mechanisms of isoprostane generation. Synthesis of prostanoids and isoprostanes, including 8-epi-PGF(2alpha), was studied under conditions favouring radical formation. Cultured glomeruli formed different prostanoids including 8-epi-PGF(2alpha). Upon LPS challenge cyclo-oxygenase (COX)-2 expression was enhanced, and this was paralleled by a 2 - 9-fold increase in prostanoid formation, including isoprostanes. Addition of COX-isoform unselective inhibitors (diclofenac, indomethacin) or a selective inhibitor (NS-398) suppressed the synthesis of prostanoids, 8-epi-PGF(2alpha) and total isoprostane fraction; however, inhibition of the latter was less pronounced. Antioxidants such as butylated hydroxytoluene (BHT), nordihydroguaiaretic acid (NDGA), or dimethylurea exhibited an only minimal inhibitory effect on 8-epi-PGF(2alpha) synthesis. Moreover, ROS-generating drugs (menadione, methylviologen) or NADPH-driven radical formation were unable to cause the generation of significant amounts of 8-epi-PGF(2alpha) by rat glomeruli. In contrast, the total isoprostane fraction could be increased by menadione addition. These data provide further evidence for a radical-independent, but COX-dependent formation of 8-epi-PGF(2alpha) in renal tissue. Regarding the other isoprostanes, both radicals and COX enzymes contribute to their formation. Based on our data we assume that elevated release of vasoactive 8-epi-PGF(2alpha) has to be expected under conditions when the prostanoid system in the kidney is stimulated, e.g. under inflammatory conditions. Regarding renal oxidative injuries, the usefulness of 8-epi-PGF(2alpha) as a representative marker molecule of oxidative stress has to be questioned.     

Escape from tension induced by noradrenaline or electrical stimulation in isolated mesenteric arteries

Isolated mesenteric arteries, studied under 25-30 mg resting tension, responded to prolonged noradrenaline or electrical stimulation with a 50-500 mg increase in tension from which they subsequently escaped towards resting tension levels.