Alpha2-adrenoceptors in opossum kidney cells couple to stimulation of mitogen-activated protein kinase independently of adenylyl cyclase inhibition.

We have compared the effects of adrenaline on activation of mitogen-activated protein kinase (MAP kinase), cyclic AMP accumulation and [3H]thymidine uptake in OK cells, a cell line derived from proximal tubules of the opossum kidney. Effects of serotonin and the direct protein kinase C activator, phorbol-12-myristate-13-acetate (PMA), were also studied. Adrenaline transiently (peak at 5 min, return to baseline by 30 min) and concentration-dependently (EC50 between 10 and 100 nM) stimulated MAP kinase activity. Maximal stimulation was approximately 100% above basal and was similar to the effects of 1 microM serotonin or 1 microM PMA. MAP kinase activation by adrenaline was inhibited by 10 microM phentolamine or 1 microM yohimbine but not significantly affected by 100 nM prazosin or 200 nM pindolol. The selective alpha2-adrenoceptor agonist UK 14,304 (10 microM) also stimulated MAP kinase activity. Activation of the 42 and 44 kDa ERK forms of MAP kinase was demonstrated by immunoblot analysis. The effect of adrenaline and UK 14,304 on MAP kinase was inhibited by pertussis toxin pretreatment and by the MAP kinase kinase inhibitor, PD 98059 (100 microM). Stimulation of MAP kinase activity was independent of cellular cAMP levels and was not affected by protein kinase C downregulation. Adrenaline, UK 14,304, serotonin, and PMA stimulated [3H]thymidine uptake, an effect inhibited by PD 98059. We conclude that adrenaline stimulates MAP kinase activity in OK-cells via alpha2-adrenoceptors and pertussis sensitive G proteins. While this occurs independently of cellular cAMP levels and protein kinase C, it involves the MEKI form of MAP kinase kinase and the ERK forms of MAP kinase. This activation results in enhanced cellular proliferation as assessed by [3H]thymidine uptake.

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

1. 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. 2. NPY and its analogues, peptide YY (PYY), [Leu31, Pro34]NPY and NPY13-36, were infused in incremental doses of 0.3, 1 and 3 micrograms 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 1-5 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. 3. 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; NPY13-36 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. 4. 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. NPY13-36 did not reduce plasma renin activity. None of the peptides significantly affected plasma aldosterone concentrations. 5. In another series of experiments infusion of PYY3-36 (2 micrograms kg-1 min-1 for 120 min) did not reduce renal blood flow but significantly enhanced diuresis and natriuresis to a similar extent as the NPY 2 micrograms kg-1 min-1. 6. In a final series of experiments the Y1-selective antagonist, BIBP 3226 (1 or 10 micrograms kg-1 min-1) dose-dependently antagonized reductions of renal blood flow elicited by bolus injections of NPY (0.1-30 micrograms kg-1). BIBP 3226 (10 micrograms kg-1 min-1) also inhibited the effects of a 120 min infusion of NPY (2 micrograms 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. 7. 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.

Protein kinase C isoenzymes in rat and human cardiovascular tissues.

1. We have compared the expression of protein kinase C (PKC) activity and immuno-detectable isoenzymes in cytosolic and membrane extracts of rat and human cardiovascular tissues (heart, kidney, aorta, saphenous vein). Experiments were performed in raw extracts and upon combined diethylaminoethylcellulose (DEAE) and phenylsepharose column chromatography. 2. PKC activity that bound to DEAE mostly eluted with 200 mM NaCl. DEAE-purified PKC from all tissues except rat kidney bound almost quantitatively to phenylsepharose and eluted with 0.5-0 M NaCl. 3. Immunoblots with an antibody against classical PKCs and the activator profile for phosphatidylserine, diolein and Ca2+ revealed that the PKC from rat kidney, which did not bind to phenylsepharose, was most probably due to a proteolytically-generated, constitutively active PKC which is not under the control of a regulatory subunit. 4. Studies in the reference tissue, rat brain, demonstrated that all PKC isoenzymes investigated (classical PKCs alpha, beta, gamma, new PKCs delta, epsilon, eta, theta, and atypical PKCs zeta, lambda, iota) have similar DEAE and phenylsepharose chromatography elution profiles. In the functional assay an inhibitor of all known PKC isoenzymes, bisindolylmaleimide, and a specific inhibitor of classical PKCs, Gö 6976, both inhibited PKC from rat brain completely and with high potency indicating that the functional assay preferentially detects classical PKC isoenzymes. 5. Each PKC isoenzyme had a tissue-specific expression profile which was similar in rat and man. The classical PKC alpha, the new PKCs delta and epsilon and all atypical PKCs were detectable in most tissues, whereas the PKC beta and PKC gamma were not detected in any pheripheral tissue; PKC eta and PKC theta were found in some tissues. 6. We conclude that combined DEAE and phenylsepharose chromatography is useful to enrich and detect PKC isoenzymes; no major species differences in tissues-specific expression patterns appear to exist between rat and man.

Endothelin-induced inositol phosphate formation in rat kidney. Studies on receptor subtypes, G-proteins and regulation during ontogenesis.

The aim of this study was to characterize the properties of endothelin (ET)-receptor subtypes mediating inositol phosphate (IP)-formation in rat kidney and their regulation during ontogenesis. In renal cortical slices of adult rats (12-16 weeks old) ET's concentration-dependently increased IP-formation with an order of potency ET-1 >> ET-3. While the non-selective ET-receptor antagonist bosentan (10 microM) completely suppressed ET-induced IP-formation, the ETA-receptor antagonist BQ-123 (10 microM) inhibited it only by 70%, the ETB-receptor antagonist IRL 1038 (1 microM) by 25%; combined application of BQ-123 + IRL 1038 caused complete inhibition of ET-1-induced IP-formation. Pretreatment of isolated renal cells with pertussis toxin (PTX, 500 ng/ml) overnight did not attenuate but significantly increased ET-1-induced IP-formation. Ontogenetic studies in renal sites from neonatal, 1, 2, 3, 6, 12 and 24 weeks old rats revealed that ET-1-induced IP-formation maturation-dependently declined being highest in neonatal rats (increase: 169% over basal) and lowest in 24 weeks old rats (increase: 47% over basal). This decline in ET-induced IP-formation was accompanied by a decrease in renal ET-receptor number and the amount of immunodetectable Gq/11 (assessed by Western-blotting using the QL-antiserum). Moreover, ET-receptor subtypes changed during the maturation process: from neonates to 12 weeks old rats number and functional responsiveness of ETA-receptors declined, while that of ETB-receptors increased. We conclude that in adult rat renal cortex ET-induced IP-formation is mediated by activation of both ETA- and ETB-receptors and does not involve a PTX-sensitive G-protein. ET-induced IP-formation declines during the maturation process; this is associated with a decrease in ET-receptor number and the immunodetectable amount of Gq/11.

Neuropeptide Y-enhanced diuresis and natriuresis in anaesthetized rats is independent of renal blood flow reduction.

1. Neuropeptide Y (NPY) has been reported to enhance diuresis and natriuresis in anaesthetized rats although it is a potent renal vasoconstrictor in vitro in vivo in several species. Therefore, we have investigated anaesthetized rats to see whether reduction in renal blood flow (RBF) and enhancement of diuresis and natriuresis can occur concomitantly, and how diuresis and natriuresis might be enhanced despite reduced RBF. 2. Systemic or intrarenal NPY infusion (0.03-3 micrograms kg-1 min-1) had only a small effect on mean arterial pressure (maximal increase 15-20 mmHg) and heart rate (maximal decrease 30 beats min-1) but dose-dependently reduced RBF (maximal peak reduction 3 ml min-1) Endogenous creatinine clearance was not significantly altered. 3. In anaesthetized rats systemic infusion of 1 or 3 micrograms kg-1 min-1 NPY enhanced urine formation and sodium and calcium excretion by a maximum of 110, 110 and 45%, respectively, but did not alter potassium excretion. Enhancement of diuresis was also detectable in conscious rats. 4. The diuretic and natriuretic effects of systemically infused NPY were at least partly maintained in rats with decapsulated kidneys and in rats where NPY-induced increase of renal perfusion pressure was excluded mechanically by an adjustable clamp placed on the abdominal aorta. 5. Intrarenal infusion of 0.3 or 1 microgram kg-1 min-1 NPY reduced RBF to a greater extent than systemic infusion (maximal peak reduction 4 ml min-1) but caused a smaller enhancement or even a reduction of urine formation and sodium excretion. 6. We conclude that systemic infusion of NPY reduces RBF by a direct effect on the renal vasculature. Systemic NPY infusion enhances urine formation and sodium and calcium excretion. This occurs independently (at least in part) of pressure natriuresis by formation and/or release of an extrarenal factor which might act on distal tubules and/or collecting ducts.

Is the glycogen synthase analogue C1-peptide a suitable fluorescent substrate for routine measurements of protein kinase C?

We have compared a new commercially available non-radioactive protein kinase C (PKC) activity assay based on the fluorescent [A9,10K11]glycogen synthase1-11 analogue C1-peptide with a classical radioactive assay based on myelin basic protein4-14 (MBP4-14) and other substrates. The C1-peptide had lower affinity for PKC from rat brain than substrates such as MBP4-14, [S25]PKC alpha 19-31, and [A9,10K11,12]glycogen synthase1-12. The sensitivity of the C1-peptide-based assay was considerably lower than that of the MBP4-14-based assay. The C1-peptide was readily degraded in an ATP-independent manner by crude and DEAE-column chromatography-purified cytosolic extracts from rat brain, rat kidney, SK-N-MC and L929 cells. In rat kidney this degradation was not prevented by many common protease inhibitors. Phenylsepharose column chromatography separated the C1-peptide degrading activity from PKC. We conclude that the C1-peptide-based fluorescent PKC assay is applicable to highly purified PKC preparations but has low sensitivity and is not applicable to crude extracts due to substrate degradation.

Does [3H]2-methoxy-idazoxan (RX 821002) detect more alpha-2-adrenoceptor agonist high-affinity sites than [3H]rauwolscine? A comparison of nine tissues and cell lines.

The authors compared [3H]2-methoxy-idazoxan (RX 821002) and [3H]rauwolscine binding in rat cerebral cortex, spleen and kidney; guinea pig kidney; porcine kidney; human kidney and platelets and HEL and NG 108-15 cells. [3H]RX 821002 had less nonspecific binding and higher affinity than [3H]rauwolscine in most models. Although both ligands detected similar alpha-2 adrenoceptor numbers in rat, porcine and human kidney and in NG 108-15 cells in saturation experiments, [3H]RX 821002 detected more alpha-2 adrenoceptors than [3H]rauwolscine in rat cerebral cortex and spleen, guinea pig kidney, human platelets and HEL cells. These differences were seen in Tris and in glycylglycine buffer regardless of whether EDTA, MgCl2, MgCl2 plus GTP or GTP plus NaCl was added to the former and were not explained by additional labeling of serotonin or dopamine receptors or nonadrenergic sites; in contrast, [3H]rauwolscine also labeled nonadrenergic sites in porcine kidney. In prazosin competition experiments, both ligands differentially recognized alpha-2-adrenoceptor subtypes but this could not account for the observed differences in detected receptor numbers. In epinephrine competition experiments, both ligands labeled similar numbers of agonist low affinity sites in all models; [3H]RX 821002, however, labeled more agonist high-affinity sites than [3H]rauwolscine did in models in which it detected a greater total number of receptors. It was concluded that [3H]RX 821002 is a more suitable ligand for the detection of alpha-2 adrenoceptor than [3H]rauwolscine because of less nonspecific binding, higher affinity and greater specificity for alpha-2 adrenoceptors; moreover, [3H]rauwolscine appears not to detect all agonist high-affinity sites of alpha-2 adrenoceptors.

Comparison of alpha 1A- and alpha 1B-adrenoceptor coupling to inositol phosphate formation in rat kidney.

We have compared the coupling mechanisms of rat renal alpha 1A- and alpha 1B-like adrenoceptors to inositol phosphate formation. The experiments were performed in parallel in native renal tissue preparations and in those where alpha 1B-adrenoceptors had been inactivated by treatment with 10 mumol/l chloroethylclonidine for 30 min at 37 degrees C; renal slices were used in most experiments but isolated renal cells were also used in some cases. The Ca2+ chelating agent, EGTA (5 mmol/l), reduced noradrenaline-stimulated inositol phosphate formation in native but enhanced it in chloroethylclonidine-treated renal slices. The inhibitory effect of EGTA was not mimicked by 100 nmol/l nifedipine. Inactivation of 87% of cellular Gi by 16-20 h treatment with 500 ng/ml pertussis toxin did not significantly affect noradrenaline-stimulated inositol phosphate formation in isolated renal cells but abolished the inhibitory effect of chloroethylclonidine. The adenylate cyclase activator, forskolin (20 mumol/l), inhibited noradrenaline-stimulated inositol phosphate formation in native and chloroethylclonidine-treated slices, and the inhibitory effects of chloroethylclonidine treatment and forskolin were additive. We conclude that in rat kidney inositol phosphate formation via alpha 1B-like adrenoceptors may involve the influx of extracellular Ca2+ and a pertussis toxin-sensitive G-protein but is insensitive to inhibition by forskolin. In contrast alpha 1A-like adrenoceptor-mediated inositol phosphate formation does not require the presence of extracellular Ca2+ or of Gi and is sensitive to inhibition by forskolin. In comparison to published data from other model systems we further conclude that the signaling mechanisms of alpha 1-adrenoceptor subtypes may depend on their cellular environment.

Ontogenesis of sympathetic responsiveness in spontaneously hypertensive rats. II. Renal G proteins in male and female rats.

Previously we have reported an increased renal alpha 1- and beta-adrenergic receptor expression in male spontaneously hypertensive rats that occurred ontogenetically in parallel with blood pressure elevation. However, increased receptor numbers were not accompanied by enhanced stimulation of inositol phosphate and cyclic AMP formation, respectively, indicating relative desensitization. We have now quantified alpha-subunits of the G proteins Gs (Gs short and Gs long), G(i), and Gq by immunoblotting and pertussis toxin-catalyzed ADP-ribosylation in renal membranes from 3-, 6-, 8-, and 28-week-old normotensive and spontaneously hypertensive male Wistar-Kyoto rats; additionally, 28-week-old female normotensive and spontaneously hypertensive rats were studied. During ontogenesis of male normotensive rats, Gs short increased, Gs long remained unchanged, and G(i) alpha and Gq alpha decreased. In adult normotensive rats no sex differences were detected for Gs short, Gs long, and G(i) alpha. When male rats from the normotensive and spontaneously hypertensive strains were compared, all G protein alpha-subunits were similar in the prehypertensive phase (3 weeks). In established hypertension (28 weeks), Gs long and Gq alpha were reduced, whereas Gs short and G(i) alpha remained unchanged. Gs long was also reduced during the development of hypertension (6 and 8 weeks), whereas Gs short and G(i) alpha were not consistently altered in this phase. The reduction in Gs long seen in male adult hypertensive rats was not detectable in female hypertensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Distinct effects of thioredoxin and antioxidants on the activation of transcription factors NF-kappa B and AP-1.

The transcription factors NF-kappa B and AP-1 have been implicated in the inducible expression of a variety of genes involved in responses to oxidative stress and cellular defense mechanisms. Here, we report that thioredoxin, an important cellular protein oxidoreductase with antioxidant activity, exerts different effects on the activation of NF-kappa B and AP-1. Transient expression or exogenous application of thioredoxin resulted in a dose-dependent inhibition of NF-kappa B activity, as demonstrated in gel shift and transactivation experiments. AP-1-dependent transactivation, in contrast was strongly enhanced by thioredoxin. A similar increase of AP-1 activity was also observed with other, structurally unrelated antioxidants such as pyrrolidine dithiocarbamate and butylated hydroxyanisole, indicating that the thioredoxin-induced increase of AP-1 activation was indeed based on an antioxidant effect. Moreover, the stimulatory effect on AP-1 activity was found to involve de novo transcription of the c-jun and c-fos components but to be independent of protein kinase C activation. These results suggest that thioredoxin plays an important role in the regulation of transcriptional processes and oppositely affects NF-kappa B and AP-1 activation.

Alpha 1-adrenoceptor subtype affinities of drugs for the treatment of prostatic hypertrophy. Evidence for heterogeneity of chloroethylclonidine-resistant rat renal alpha 1-adrenoceptor.

We have used radioligand binding and inositol phosphate accumulation studies to determine the affinity at mixed alpha 1A- and alpha 1B-adrenoceptors (rat cerebral cortex and kidney), alpha 1A-adrenoceptors (rat cerebral cortex and kidney following inactivation of alpha 1B-adrenoceptors by chloroethylclonidine treatment) and alpha 1B-adrenoceptors (rat spleen) for drugs currently under investigation for the treatment of benign prostatic hypertrophy, alfuzosin, naftopidil and (-)- and (+)-tamsulosin. Alfuzosin and naftopidil had similar affinities in all model systems (approximately 10 nM and 130 nM, respectively) and lacked relevant selectivity for alpha 1-adrenoceptor subtypes. Their potency to inhibit noradrenaline-stimulated inositol phosphate formation in cerebral cortex matched their affinities as determined in the binding studies. Tamsulosin had higher affinity at alpha 1A- than at alpha 1B-adrenoceptors, and was slightly more potent than alfuzosin and naftopidil at alpha 1B- and considerably more potent at alpha 1A-adrenoceptors. However, the interaction of the tamsulosin isomers with chloroethylclonidine-insensitive (alpha 1A-like) adrenoceptors was complex. A detailed analysis of the tamsulosin data and those obtained with other drugs, most notably noradrenaline and oxymetazoline, suggested that chloroethylclonidine-insensitive alpha 1-adrenoceptors may be heterogeneous and that this heterogeneity may differ between cerebral cortex and kidney of the rat.

Norepinephrine and neuropeptide Y increase intracellular Ca2+ in cultured porcine aortic smooth muscle cells.

We used the fluorescent Ca2+ indicator Fura-2 in cultured porcine aortic smooth muscle cells (PASMC) to study effects of the sympathetic neurotransmitters norepinephrine (NE) and neuropeptide Y (NPY) on free intracellular Ca2+ (Cai). Both transmitters transiently increased intracellular Ca2+ in a concentration-dependent manner. Selective agonists and antagonists demonstrated that the NE-stimulated Cai increase is predominantly (if not exclusively) mediated by alpha 2-adrenoceptors, whereas the NPY response appears to be mediated by the peptide YY-insensitive Y3-like receptor subtype. Pretreatment of cells with pertussis toxin abolished NPY and alpha-adrenoceptor agonist-stimulated intracellular Ca2+ elevations (but not those stimulated by angiotensin II) suggesting involvement of a Gi-like G-protein. alpha 2-Adrenoceptor-stimulated Ca2+ increases resulted from mobilization from intracellular stores, whereas Y3-like NPY receptors mobilized Ca2+ from intracellular stores and also promoted Ca2+ influx.

Is PP56 (D-myo-inositol-1,2,6-trisphosphate) an antagonist at neuropeptide Y receptors?

PP56 (D-myo-inositol-1,2,6,-trisphosphate) has been reported to specifically inhibit neuropeptide Y-mediated effects in vasculature, heart and brain; because of its reversible but non-competitive antagonism interaction with neuropeptide Y receptor signalling or allosteric modulation of neuropeptide Y binding have been postulated. These possibilities were tested in the present study. PP56 did not affect [125I]neuropeptide Y binding to HEL- or SK-N-MC-cells or to porcine splenic membranes. PP56 did not inhibit neuropeptide Y-stimulated Ca2+ increases in HEL- or SK-N-MC-cells or in cultured porcine aortic vascular smooth muscle cells but if anything slightly enhanced it. PP56 did not antagonize the neuropeptide Y-mediated inhibition of forskolin-stimulated cAMP accumulation in HEL-cells. We conclude that previously reported antagonistic effects of PP56 occur distal to the neuropeptide Y receptor or its second messenger systems Ca2+ and cAMP or may be restricted to neuropeptide Y receptors in certain model systems.