Angela Bischoff

Sphingosine-1-phosphate and sphingosylphosphorylcholine constrict renal and mesenteric microvessels in vitro

Sphingolipids such as sphingosine‐1‐phosphate (SPP) and sphingosylphosphorylcholine (SPPC) can act both intracellularly and at G‐protein‐coupled receptors, some of which were cloned and designated as Edg‐receptors. Sphingolipid‐induced vascular effects were determined in isolated rat mesenteric and intrarenal microvessels. Additionally, sphingolipid‐induced elevations in intracellular Ca2+ concentration were measured in cultured rat aortic smooth muscle cells. SPPC and SPP (0.1–100 μmol l−1) caused concentration‐dependent contraction of mesenteric and intrarenal microvessels (e.g. SPPC in mesenteric microvessels pEC50 5.63±0.17 and Emax 49±3% of noradrenaline), with other sphingolipids being less active. The vasoconstrictor effect of SPPC in mesenteric microvessels was stereospecific (pEC50 D‐erythro‐SPPC 5.69±0.08, L‐threo‐SPPC 5.31±0.06) and inhibited by pretreatment with pertussis toxin (Emax from 44±5 to 19±4%), by chelation of extracellular Ca2+ with EGTA and by nitrendipine (Emax from 40±6 to 6±1 and 29±6%, respectively). Mechanical endothelial denudation or NO synthase inhibition did not alter the SPPC effects, while indomethacin reduced them (Emax from 87±3 to 70±4%). SPP and SPPC caused transient increases in intracellular Ca2+ concentrations in rat aortic smooth muscle cells in a pertussis toxin‐sensitive manner. Our data demonstrate that SPP and SPPC cause vasoconstriction of isolated rat microvessels and increase intracellular Ca2+ concentrations in cultured rat aortic smooth muscle cells. These effects appear to occur via receptors coupled to pertussis toxin‐sensitive G‐proteins. This is the first demonstration of effects of SPP and SPPC on vascular tone and suggests that sphingolipids may be an hitherto unrecognized class of endogenous regulators of vascular tone.

Sphingosine-1-phosphate reduces rat renal and mesenteric blood flow in vivo in a pertussis toxin-sensitive manner

Sphingolipids such as sphingosine‐1‐phosphate (SPP) and sphingosylphosphorylcholine constrict isolated rat intrarenal and mesenteric microvessels in vitro. The present study investigates their effects on the cardiovascular system in vivo in anaesthetized rats. The animals were given intravenous or intrarenal arterial bolus injections of sphingolipids (0.1–100 μg kg−1) with subsequent measurements of mean arterial pressure, heart rate and renal and mesenteric blood flows (RBF, MBF) using a pressure transducer and electromagnetic flow probes, respectively. Intravenous injection of SPP rapidly (within 30 s), transiently and dose‐dependently reduced RBF (maximally −4.0±0.3 ml min−1) and MBF (maximally −1.4±0.2 ml min−1), without affecting mean arterial pressure or heart rate. Other sphingolipids had no significant effect. Intrarenal arterial SPP administration caused greater blood flow reductions (maximally −6.4±0.3 ml min−1) than systemic administration. Upon intrarenal administration, sphingosylphos‐ phorylcholine also lowered RBF (maximally −2.8±0.6 ml min−1), while the other sphingolipids remained without effect. Pretreatment with pertussis toxin (PTX, 10 μg kg−1) 3 days before the acute experiment abolished the SPP‐induced reductions of RBF and MBF. These data demonstrate, that SPP is a potent vasoconstrictor in vivo, particularly in the renal vasculature, while the other structurally related sphingolipids had little if any effects. The PTX‐sensitivity strongly suggests that the effects of SPP on renal and mesenteric blood flow are mediated by receptors coupled to Gi‐type G‐proteins.

Comparison of problem-and lecture-based pharmacology teaching

Problem-based learning (PBL) is gaining interest in many medical schools. Although various approaches have been labelled PBL, it remains unclear which approach is most appropriate for pharmacology courses. Moreover, some teachers remain sceptical about whether PBL is adequate to convey the numerous facts medical students need to memorize about drugs. However, comparisons of PBL methods with conventional lecture-based learning (LBL) methods within general pharmacology courses for medical students show that PBL students are at least as successful in standardized tests but enjoy their course to a greater extent than do LBL students.

Receptor subtypes Y1 and Y5 are involved in the renal effects of neuropeptide Y

Systemic infusion of neuropeptide Y (NPY) reduces renal blood flow and can concomitantly increase diuresis, natriuresis and calciuresis in anaesthetized rats. The present study was designed to investigate whether the apparently contradictory NPY effects on renal blood flow and urine formation and composition are mediated by distinct NPY receptor subtypes. NPY and its analogues, peptide YY (PYY), [Leu31, Pro34]NPY and NPY1336, were infused in incremental doses of 0.3, 1 and 3 μg kg−1 min−1 for 45 min each and the results compared to those obtained in vehicle‐infused rats. Renal blood flow was monitored in 15 min intervals, while urine excretion and composition were determined in 15 min collection periods. Plasma renin activity and aldosterone concentrations were measured at the end of the final infusion period. Relative to vehicle NPY, PYY and [Leu31, Pro34]NPY dose‐dependently reduced renal blood flow and increased diuresis, natriuresis and calciuresis with roughly similar potency; NPY1336 slightly but significantly increased renal blood flow but had no effect on diuresis, natriuresis and calciuresis. None of the peptides significantly affected endogenous creatinine clearance or kaliuresis. Plasma renin activity was significantly reduced by PYY. Quantitatively similar reductions were observed with NPY and [Leu31, Pro34]NPY but failed to reach statistical significance with the given number of experiments. NPY1336 did not reduce plasma renin activity. None of the peptides significantly affected plasma aldosterone concentrations. In another series of experiments infusion of PYY336 (2 μg kg−1 min−1 for 120 min) did not reduce renal blood flow but significantly enhancd diuresis and natriuresis to a similar extent as the NPY 2 μg kg−1 min−1. In a final series of experiments the Y1‐selective antagonist, BIBP 3226 (1 or 10 μg kg−1 min−1) dose‐dependently antagonized reductions of renal blood flow elicited by bolus injections of NPY (0.130 μg kg−1). BIBP 3226 (10 μg kg−1 min−1) also inhibited the effects of a 120 min infusion of NPY (2 μg kg−1 min−1) on renal blood flow but had only minor inhibitory effects on enhancements of diuresis and did not significantly affect enhancements of natriuresis. We conclude that NPY reduces renal blood via a classical Y1 subtype of NPY receptor. In contrast enhancements of diuresis, natriuresis and calciuresis occur via a distinct subtype which resembles the receptor that mediates NPY‐induced enhancement of food intake.

Emerging functions for neuropeptide Y5 receptors

The Y5 subtype of neuropeptide Y (NPY) receptors has raised considerable interest as a mediator of NPY-stimulated food intake, but with the advent of recent data, this hypothesis has come into question. Moreover, Y5 receptor-selective drugs might not be specific for food intake because additional functions in the central and peripheral nervous systems, including endogenous anti-epileptic activity, attenuation of morphine withdrawal symptoms, enhancement of diuresis and natriuresis, lowering of blood glucose and reduction of acetylcholine release in the ileum, have recently been reported to occur via Y5-like receptors. Given that mRNA for the cloned Y5 receptor is apparently restricted to the CNS, Angela Bischoff and Martin Michel discuss the possible existence of additional NPY receptor subtypes with Y5-like recognition features and their presence in peripheral tissues.

Lysosphingolipid receptor-mediated diuresis and natriuresis in anaesthetized rats

Lysosphingolipids such as sphingosine‐1‐phosphate (SPP) and sphingosylphosphorylcholine (SPPC) can act on specific G‐protein‐coupled receptors. Since SPP and SPPC cause renal vasoconstriction, we have investigated their effects on urine and electrolyte excretion in anaesthetized rats. Infusion of SPP (1 – 30 μg kg−1 min−1) for up to 120 min dose‐dependently but transiently (peak after 15 min, disappearance after 60 min) reduced renal blood flow without altering endogenous creatinine clearance. Nevertheless, infusion of SPP increased diuresis, natriuresis and calciuresis and, to a lesser extent, kaliuresis. These tubular lysosphingolipid effects developed more slowly (maximum after 60 – 90 min) and also abated more slowly upon lysosphingolipid washout than the renovascular effects. Infusion of SPPC, sphingosine and glucopsychosine (3 – 30 μg kg−1 min−1 each) caused little if any alterations in renal blood flow but also increased diuresis, natriuresis and calciuresis and, to a lesser extent, kaliuresis. Pretreatment with pertussis toxin (10 μg kg−1 3 days before the acute experiment) abolished the renovascular and tubular effects of 30 μg kg−1 min−1 SPP. These findings suggest that lysosphingolipids are a hitherto unrecognized class of endogenous modulators of renal function. SPP affects renovascular tone and tubular function via receptors coupled to Gi‐type G‐proteins. SPPC, sphingosine and glucopsychosine mimic only the tubular effects of SPP, and hence may act on distinct sites.

Alpha2B-adrenoceptors couple to Ca2+ increase in both endogenous and recombinant expression systems

The ability of cloned human alpha2B-adrenoceptors heterologously expressed in Sf9 cells and endogenous alpha2B-adrenoceptors in NG 108-15 neuroblastoma x glioma cells to couple to increase of intracellular Ca2+ was studied. Ca2+ increases in NG 108-15 cells were detectable but slight, whereas those in alpha2B-adrenoceptor-expressing Sf9 cells were greater. In the latter, the maximum Ca2+ increase correlated positively, and the EC50-value of noradrenaline negatively, with the receptor expression density. The order of potency of the agonists was D-medetomidine ([D]-4-[5]-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole) > noradrenaline approximately = clonidine > oxymetazoline, with clonidine and UK14,304 (5-bromo-N-[4,5-dihydro-1H-imidazole-2-yl]-6-quinoxalinamine) being weak partial agonists. In Sf9 cells Ca2+ increases consisted of concomitant mobilization from an intracellular store and influx of extracellular Ca2+. In these cells alpha2B-adrenoceptor stimulation also increased the inositol 1,4,5-trisphosphate mass. We conclude that alpha2B-adrenoceptors can couple to intracellular Ca2+ increases which may involve prior activation of phospholipase C.

Transient relaxation of rat mesenteric microvessels by ceramides

We have investigated the vasodilating effects of D‐erythro‐C2‐ceramide (C2‐ceramide) in methoxamine‐contracted rat mesenteric microvessels. C2‐ceramide (10–100 μM) caused a concentration‐dependent, slowly developing relaxation which reached maximum values after ∼10 min and partially abated thereafter. Endothelium removal or inhibitors of guanylyl cyclase (3 μM ODQ), protein kinase A (10 μM H7, 1 μM H89) and various types of K+ channels (10 μM BaCl2, 3 mM tetraethylammonium, 30 nM charybdotoxin, 30 nM iberiotoxin, 300 nM apamine, 10 μM glibenclamide) had only small if any inhibitory effects against C2‐ceramide‐induced vasodilation, but some of them attenuated vasodilation by sodium nitroprusside or isoprenaline. A combination of ODQ and charybdotoxin almost completely abolished C2‐ceramide‐induced vasodilation. A second administration of C2‐ceramide caused a detectable but weaker relaxation. L‐threo‐C2‐ceramide (100 μM), which should not be a substrate to ceramide metabolism, had no biphasic time course. The ceramidase inhibitor (1S,2R)‐D‐erythro‐2‐(N‐myristoylamino)‐1‐phenyl‐1‐propanol (100 μM) alone caused some vasodilation, indicating vasodilation by endogenous ceramides, and also hastened relaxation by exogenous C2‐ceramide. The late‐developing reversal of C2‐ceramide‐induced vasodilation was absent when α‐adrenergic tone was removed by addition of 10 μM phentolamine. We conclude that C2‐ceramide relaxes rat resistance vessels in an endothelium‐independent manner which is prevented only by combined inhibition of guanylyl cyclase and charybdotoxin‐sensitive K+ channels. The vasodilation abates with time partly due to desensitization of the ceramide response and partly due to metabolism of C2‐ceramide to an inactive metabolite.

Limited signal transduction repertoire of human Y5 neuropeptide Y receptors expressed in HEC-1B cells

In HEC-1B cells transfected with human Y(5) neuropeptide Y (NPY) receptors (but not in non-transfected cells) NPY inhibited forskolin-stimulated cAMP accumulation in a pertussis toxin-sensitive manner (-log EC(50) 8.88 +/- 0.25). Elevations of intracellular Ca(2+) were largely restricted to very high NPY concentrations and similar in transfected and nontransfected cells. NPY did not increase inositol phosphate accumulation and did not activate a variety of isoforms of protein kinase C or mitogen-activated protein kinases. We conclude that at least upon expression in HEC-1B cells the signal transduction of Y(5) NPY receptors is limited to inhibition of cAMP accumulation.

The Y1 antagonist BIBP 3226 inhibits potentiation of methoxamine-induced vasoconstriction by neuropeptide Y

We investigated the interaction of neuropeptide Y (NPY) with the α1-adrenoceptor agonist, methoxamine, in control of mean arterial pressure, renovascular resistance and mesenteric vascular resistance in anaesthetized rats. Infusion of 3.0 but not 0.3μg/kg/min NPY enhanced the elevations of all three haemodynamic parameters caused by bolus injections of methoxamine (10–100μg/kg). These enhancements largely involved a prolongation of the methoxamine effects. While infusion of the Y1 NPY receptor-selective antagonist, BIBP 3226 (10μg/kg/min), alone did not alter methoxamine-induced vasoconstriction, it inhibited the potentiation by NPY. We conclude that NPY can potentiate methoxamine-induced vasoconstriction in vivo. This is mediated predominantly, if not exclusively, via the Y1 receptor. Endogenously released NPY does not appear to reach sufficient concentrations to cause tonic systemic vasoconstriction or potentiation thereof in the anaesthetized rat.