Differential effect of DPI 201-106 on the sensitivity of the myofilaments to Ca2+ in intact and skinned trabeculae from control and myopathic human hearts.

Activation of protein kinase C (PKC) and elevation of intracellular calcium ion concentration ([Ca++]i) result from phosphatidylinositol biphosphate (PIP2) breakdown. We previously demonstrated that PKC activation inhibits arginine vasopressin (AVP)-induced osmotic water flow in rabbit cortical collecting tubule (CCT) perfused in vitro at 37 degrees C. To estimate the potential significance of PIP2 turnover as a modulator of water transport in this nephron segment, we examined the effect of Ca on AVP action and explored the mechanisms of action of PKC and increased [Ca++]i. In rabbit CCTs perfused at 37 degrees C, pretreatment with bath A23187 (2 x 10(-8) M, 2 x 10(-6) M), a Ca ionophore, almost totally suppressed AVP (10 microU/ml)-induced peak hydraulic conductivity (Lp). The suppression by 2 x 10(-8) M A23187 was as potent as that by 2 x 10(-6) M A23187, and significant even when it was administered 10 min after AVP. When phorbol myristate acetate (PMA, 10(-9) M), a PKC activator, and A23187 (2 x 10(-8) M) were placed in the bath simultaneously, the combined suppressive effect on peak Lp was greater than that of either inhibitor alone. However, the mechanisms of inhibition by PMA and A23187 were different. While both 10(-7) and 10(-9) M PMA suppression are primarily post-cAMP, A23187 predominantly suppressed a pre-cAMP step: 10(-4) M chlorophenylthio-cAMP-induced peak Lp was not affected by 2 x 10(-8) M A23187, and only partially inhibited by 2 x 10(-6) M A23187. The PMA (10(-7) M) suppression of AVP-induced peak Lp was totally reversed by bath staurosporine (10(-7) M), a PKC inhibitor, but not attenuated by either bath indomethacin (5 x 10(-6) M) or low Ca (1-2 x 10(-6) M) bath medium. In contrast, the A23187 (2 x 10(-8) M) suppression of the peak Lp was not affected by staurosporine, but was significantly reversed by indomethacin or low Ca bath medium. We conclude: (a) Elevation of [Ca++]i, as well as activation of PKC, suppresses the hydroosmotic effect of AVP on CCT at 37 degrees C. (b) When stimulated simultaneously these two intracellular mediators are additive in their antagonism of AVP action. These results suggest that stimulated PIP2 breakdown may be an important modulator of water transport in CCT. (c) Different mechanisms underlie PKC and Ca-mediated suppression of the AVP-induced water transport. The inhibition of AVP action by increased [Ca++]i is primarily pre-cAMP, and involves a cyclooxygenase metabolite(s) of arachidonic acid, while the inhibition by PKC is post-cAMP, and independent of cyclooxygenase products of arachidonic acid.     

Phorbol myristate acetate, dioctanoylglycerol, and phosphatidic acid inhibit the hydroosmotic effect of vasopressin on rabbit cortical collecting tubule.

We explored the role for protein kinase C (PKC) in modulating vasopressin (AVP)-stimulated hydraulic conductivity (Lp) in rabbit cortical collecting tubule (CCT) perfused in vitro at 37 degrees C. In control studies, 10 microU/ml AVP increased Lp (mean +/- SE, X 10(-7) centimeters/atmosphere per second) from 4.4 +/- 0.9 to 166.0 +/- 10.4. Pretreatment with dioctanoylglycerol (DiC8) suppressed AVP stimulated peak Lp (peak Lp, 21.9 +/- 3.1). Pretreatment with 10(-9) and 10(-7) M 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) also blocked the increase in Lp in a dose-dependent fashion (peak Lp, 59.3 +/- 7.5 and 18.6 +/- 4.8, respectively). Inactive phorbol ester, 4 alpha-phorbol 12 beta,13 alpha-didecanoate (10(-7) M), had no effect. PMA also suppressed the increase in Lp induced by 10(-4) M 8-p-chlorophenylthio-cyclic AMP (CcAMP): peak Lp was 169.4 +/- 14.9 in control, 79.2 +/- 5.5 with 10(-9) M PMA, and 25.7 +/- 2.9 with 10(-7) M PMA. Furthermore, when 10(-7) M PMA was added to the bath 10 min after exposure to AVP, the Lp response to AVP was blocked. Peak Lp was 52.4 +/- 9.6 with PMA vs. 165.1 +/- 10.0 in control. Phosphatidic acid (PA), which is thought to stimulate phosphatidylinositol (PI) turnover, produced similar inhibitory effects on AVP as well as CcAMP-stimulated Lp: PA suppressed 10-microU/ml AVP-induced peak Lp from a control value of 159.6 +/- 7.9 to 88.9 +/- 15.8, and 10(-4) M CcAMP induced peak Lp from 169.4 +/- 14.9 to 95.5 +/- 7.7. We conclude that PMA, at concentrations known to specifically activate PKC, suppresses the hydroosmotic effect of AVP on CCT; This suppression is primarily a post-cAMP event; Inhibition of AVP-stimulated Lp by DiC8 and PA also suggests an inhibitory role for the PKC system; The ability of pre- and post-AVP administration of PMA to blunt the AVP response suggests that agents that act through modulation of PI turnover in CCT may regulate the hydroosmotic effect of AVP.     

Luminal vasopressin modulates transport in the rabbit cortical collecting duct.

We explored the action of luminal AVP in rabbit CCD perfused in vitro at 37 degrees C. Nanomolar concentrations of luminal AVP induced a sustained hyperpolarization of transepithelial voltage (Vt) in contrast to a transient hyperpolarization caused by basolateral AVP. 10 microM basolateral ouabain abolished the latter but not the former change in Vt. Despite a sustained hyperpolarization (from -20.7 +/- 2.9 to -34.1 +/- 4.7 mV; P less than 0.01), 10 nM luminal AVP only slightly altered net Na+ and K+ fluxes (7.6% stimulation and no significant change, respectively). Instead, luminal AVP appeared to modulate an acetazolamide-sensitive electrogenic ion transport because 200 microM basolateral acetazolamide suppressed the luminal AVP-induced hyperpolarization (percentage of Vt from -50.4 +/- 10.8 to -5.1 +/- 1.4; P less than 0.005). In terms of water transport, 10 nM luminal AVP did not change hydraulic conductivity (Lp, x 10(-7) cm/atm per s) (from 3.9 +/- 0.8 to 5.0 +/- 1.2), but suppressed the increase in Lp induced by 20 pM basolateral AVP (134.9 +/- 19.2 vs. 204.3 +/- 21.1 in control; P less than 0.05). These findings demonstrate distinct luminal action of AVP, suggesting amphilateral regulation of epithelial transport by AVP in the CCD.     

Cytochrome P450 metabolites of arachidonic acid are potent inhibitors of vasopressin action on rabbit cortical collecting duct.

AA is metabolized by a cytochrome P450, NADPH-dependent epoxygenase to four regioisomeric epoxyeicosatrienoic acids (EETs). The EETs are further hydrated enzymatically to their respective diols, vic-dihydroxyeicosatrienoic acids (DHETs). We studied the effect of pretreatment with DHETs on 10 microU/cm2 arginine vasopressin (AVP)-stimulated hydraulic conductivity (Lp) (Lp x 10(-7) cm/atm/s, mean +/- SE) in rabbit cortical collecting ducts (CCDs) perfused in vitro at 37 degrees C. At 10(-6) M all four DHETs were potent inhibitors of the hydroosmotic effect of AVP. 14,15-DHET was the most potent isomer; it reduced AVP-induced Lp from a control value of 234.75 +/- 11.7, n = 17, to a value of 95.2 +/- 8.39, n = 5, P less than 0.0001, a reduction of AVP-mediated water flow of 60%. The inhibitory effect of 14,15-DHET was dose dependent and significant to nanomolar concentrations. 14,15-DHET at 10(-7) M was as potent an inhibitor of AVP's activity as was 10(-7) M PGE2. AVP's hydroosmotic effect is mediated through its intracellular second messenger, cAMP. 8-p-Chlorophenylthio-cAMP (CcAMP) at 10(-4) M induced a peak Lp of 189.6 +/- 11.0, n = 8; pretreatment with 10(-6) M 14,15-DHET reduced CcAMP-peak Lp to 132.0 +/- 13.4, n = 5, P less than 0.01, demonstrating a post-cAMP effect. Gas chromatography/mass spectroscopy suggests that EETs are present in extracts purified from CCDs. We conclude that cytochrome P450 epoxygenase eicosanoids are potent inhibitors of the hydroosmotic effect of vasopressin and are endogenous constituents of normal CCDs, the major target tissue for AVP.     

Vasopressin stimulates Cl- transport in ascending thin limb of Henle's loop in hamster.

The effect of arginine vasopressin (AVP) on NaCl transport was investigated in the isolated microperfused hamster ascending thin limb of Henle's loop by measuring transepithelial voltage (Vt) and transmural 22Na+ and 36Cl- fluxes. In the presence of a transmural NaCl concentration gradient (100 mM higher in the lumen), Vt was 8.4 +/- 0.4 mV. Addition of 1 nM AVP to the basolateral solution increased Vt to 9.6 +/- 0.4 mV, which corresponds to an increase in the Cl- to Na+ permselectivity ratio (PCl/PNa) from 2.8 +/- 0.2 to 3.4 +/- 0.2. AVP at physiological concentrations increased Vt in a dose-dependent manner with an ED50 of 5 pM. AVP increased the Cl- efflux coefficient from 99.6 +/- 6.3 to 131.4 +/- 10.6 x 10(-7) cm2/s without affecting the Na+ efflux coefficient. 5-Nitro-2-(3-phenyl-propylamino)-benzoate (0.2 mM), a Cl- channel inhibitor, in the perfusate decreased the basal Cl- efflux coefficient and inhibited the AVP-induced increase in this parameter. The AVP-induced increase in Vt was not affected by [d(CH2)5(1),O-Me-Tyr2,Arg8] vasopressin, a V1 receptor antagonist, but was abolished by [d(CH2)5,D-Ile2,Ile4,Arg8] vasopressin, a V2 receptor antagonist. The selective V2 agonist dDAVP in 1 nM also increased Vt from 8.6 +/- 0.7 to 9.5 +/- 0.6 mV. Dibutyryl cAMP and forskolin both increased Vt, whereas H89, an inhibitor of cAMP-dependent protein kinase, abolished the AVP-induced increase in Vt. These results demonstrate that AVP stimulates Cl- transport in the ascending thin limb of Henle's loop by activating Cl- channels via a signal transduction cascade comprising V2 receptors, adenylate cyclase, and cAMP-dependent protein kinase. The ascending thin limb of Henle's loop thus participates in the formation of concentrated urine as one of the target renal tubular segments of AVP.     

Dexamethasone inhibits epidermal growth factor-stimulated gastric epithelial cell proliferation

Epidermal growth factor (EGF) is essential to heal gastric ulcers, whereas glucocorticoid delays rat gastric ulcer healing. We found that dexamethasone inhibited EGF-stimulated rat gastric epithelial cell (RGM-1) proliferation by cell count and DNA synthesis analysis of flow cytometry and attempted to elucidate the possible mechanistic pathway via Western blot analysis. EGF (10 ng/ml) treatment for 24 h significantly increased RGM-1 cell proliferation, and dexamethasone (10(-8) and 10(-6) M) markedly suppressed EGF-stimulated cell proliferation. Western blotting results demonstrated that the phosphorylated extracellular signal-regulated kinase (pERK) (pERK1/pERK2) significantly increased at 10 min after EGF treatment. This was followed by increase of cyclooxygenase (COX)-2 expression at 3 h after EGF treatment. The continued increase of COX-2 (up to 18 h) resulted in increased intracellular prostaglandin E(2) and cyclin D1 expression significantly after 8 and 12 h of EGF treatment. Dexamethasone substantially reduced EGF-stimulated COX-2 expression at 3 and 6 h and cyclin D1 expression at 8 and 12 h. Pretreatment of RGM-1 cells with dexamethasone or 2'-amino-3'-methoxyflavone (PD98059)-mitogen-activated protein kinase kinase inhibitor (5 x 10(-5) M) significantly reduced EGF-stimulated pERK1/pERK2 expression. Simultaneous treatment of RGM-1 cells with PD98059 and EGF also markedly decreased EGF-stimulated COX-2 expression at 6 h. These findings indicate that dexamethasone significantly suppresses EGF-stimulated gastric epithelial cell proliferation, and one of the pathways involved is via inhibiting activation of ERK1/ERK2, followed by inhibition of COX-2, cyclin D1 expression, and finally DNA synthesis.     

Mechanisms of 5-hydroxytryptamine(2A) receptor activation of the mitogen-activated protein kinase pathway in vascular smooth muscle

5-Hydroxytryptamine (5-HT) activates the extracellular signal-regulated kinase (Erk) mitogen-activated protein kinases (MAPKs) in the vasculature, resulting in contraction. The mechanisms by which this occurs are unclear. G protein-coupled receptors can activate Erk MAPK pathways through a variety of mechanisms, including stimulation of Src, phosphoinositide-3 kinase (PI-3-K), protein kinase C (PKC), or the epidermal growth factor (EGF) receptor tyrosine kinase. We hypothesize that 5-HT uses one or more of these pathways. In isolated strips of rat aorta, the MAPK/Erk kinase inhibitor U0126 (50 microM), Src inhibitor PP1 (0.5 microM), PKC inhibitors calphostin C (1 microM) and chelerythrine (10 microM), and the PI-3-K inhibitor LY294002 (1-20 microM) reduced 5-HT-induced contraction. The EGF receptor tyrosine kinase inhibitor AG1478 (0.25-1 microM) was without effect. Thus, 5-HT activates PKC, Src, and possibly PI-3-K to result in contraction. In rat aortic myocytes, 5-HT (1 microM) activated Erk MAPK proteins 2- to 3-fold over basal values; activation was reduced by U0126, PP1, and LY294002 and unaffected by calphostin C or chelerythrine, wortmannin, or AG1478. The lack of effect of EGF receptor tyrosine kinase and PI-3-K inhibitors was confirmed in that the EGF receptor immunoprecipitated from 5-HT-exposed cells did not display an increase in autophosphorylation, nor did 5-HT significantly increase activation of Akt/protein kinase B, a downstream substrate for PI-3-K. These data suggest that the rat aortic 5-HT(2A) receptor uses Src but not PKC, PI-3-K, or the EGF receptor tyrosine kinase in stimulating Erk MAPK activation.     

Inhibitory effects of aminoglycosides on renal protein phosphorylation by protein kinase C

Aminoglycosides such as neomycin are commonly prescribed antibiotics; however, there is associated serious damage to the kidney. We examined the effect of aminoglycoside antibiotics on renal protein phosphorylation and found that neomycin selectively inhibited Ca++-activated, phospholipid-dependent phosphorylation of 88-kDa protein in cell lysates of the rabbit kidney. Fifty percent inhibition of phosphorylation of this protein occurred with 5 X 10(-5) to 1 X 10(-4) M neomycin. In living PtK2 cells, neomycin dose-dependently inhibited 12-O-tetradecanoyl-phorbol-13-acetate-induced phosphorylation of 88 K Da protein. This drug also inhibited phosphorylation of exogenous protein catalyzed by protein kinase C, isolated from rabbit kidney in vitro. In contrast, neomycin had little or no inhibitory effect on cyclic GMP-dependent protein kinase, cyclic AMP-dependent protein kinase, casein kinase I, casein kinase II and Ca++-calmodulin-dependent myosin light chain kinase. Whereas activity of protein kinase C was inhibited 65% by neomycin (0.1 mM) at pH 5 to 7, inhibition decreases to 33% at pH 8 and to zero at pH 9. The potencies of a series of aminoglycoside antibiotics to inhibit the kinase agreed well with number of ionizable amino groups of compounds (gamma = 0.99) and this also approximates their known nephrotoxic potential; amikacin less than or equal to kanamycin less than gentamycin less than or equal to tobramycin less than neomycin. As aminoglycoside antibiotics present in the kidney after administration of toxicological doses (10(-2) M) will inhibit the effects of protein kinase C, the aminoglycoside antibiotics-induced nephrotoxicity is discussed in relation to inhibition of intracellular protein kinase C.     

Lipoprotein of the Density 1. 006–1. 020 in the Plasma of Patients with Type III Hyperlipoproteinaemia in the Postabsorptive State

Using a rate zonal ultracentrifugal technique in 10 patients with type III hyperlipoproteinaemia, very low density lipoproteins (VLDL) (d less than 1.006, S(f) greater than 20), an intermediate lipoprotein density class with a S(f) range of 15 - 20 (Lp III), and low density lipoproteins (LDL) (S(f) 6 - 8) could be isolated from the plasma. The Lp III was found as a symmetrical peak between VLDL and LDL; its plasma concentration was 248 mg/100 ml plasma (mean), 92 - 469 mg/100 ml plasma (range); the Lp III/LDL ratio was 0.65 (mean), 0.25 - 2.3 (range); the chemical composition w/w was 35.8 percent total cholesterol, 19.3 percent triglycerides, 24.3 percent phospholipids, and 20.4 percent protein (mean). In agarose gel electrophoresis, the Lp III migrated faster than LDL, but slower than VLDL. By means of a double immunodiffusion technique and immunoelectrophoresis, apo-Lp B and apo-Lp C were detectable. In an angle head rotor at 1.4 x 10-8 g. min. Lp III can be separated from VLDL at a solvent density of 1.006, and from LDL at a density of 1.025. In the postabsorptive state in one patient with type I hyperlipoproteinaemia, five with type II and 20 with type IV, and in 50 healthy persons of both sexes, this Lp III peak was not found. The finding of a high concentration of Lp III and a rather low concentration of LDL in the plasma of patients with type III hyperlipoproteinaemia can be regarded as further evidence for an impaired catabolism of VLDL to LDL at Lp III ("intermediate" lipoprotein).     

Evidence for differential regulation of renal proximal tubular p-aminohippurate and sodium-dependent dicarboxylate transport.

In renal proximal tubules, the basolateral organic anion [p-aminohippurate (PAH)] transporter is functionally coupled to the sodium-dependent dicarboxylate transporter. This study was undertaken to elucidate whether protein kinases differentially modulate the activities of these transporters. In isolated S(2) segments of proximal tubules microdissected from rabbit kidneys, we investigated whether the transporters are regulated by tyrosine kinases, phosphatidylinositol 3-kinase (PI3K), and mitogen-activated protein kinase (MAPK). The tubules were collapsed; hence, tubular uptake of the marker substances [(3)H]PAH and [(14)C]glutarate reflects transport across the basolateral cell membrane. Genistein, a selective inhibitor of tyrosine kinase, diminished PAH uptake at 10(-7) M by 15.6 +/- 11.7% and at 10(-6) M by 25.6 +/- 9.1%. An inactive analog of genistein, diadzein, was without effect even at a concentration 100-fold higher than the lowest concentration of genistein, which produced significant reduction of PAH uptake. At 10(-7) M, wortmannin, a selective inhibitor of PI3K, reduced PAH uptake by 24.1 +/- 11.3% and, at 10(-6) M, it reduced it by 32.9 +/- 11.8%. The selective inhibitor of MAPK, PD98059, diminished PAH uptake at 5 x 10(-5) M by 23.2 +/- 6.8% and at 10(-4) M by 18.3 +/- 5.2%. Glutarate uptake was not reduced by any of these protein kinase inhibitors. Insulin had no effect on PAH uptake. These findings indicate that, in addition to protein kinase A, protein kinase C and calcium/calmodulin-dependent protein kinase II (former studies from this laboratory), as well as tyrosine kinases, PI3K, and MAPK, modulate renal basolateral PAH transport, whereas none of these protein kinases affects basolateral glutarate transport. Thus, the results provide evidence for differential regulation of basolateral transporters for PAH and dicarboxylates.     

Inhibition of airway smooth muscle tone by a phorbol ester in the guinea pig trachea: role of epithelium and receptor reserve of the contractile agent

Bronchial hyperresponsiveness in patients with asthma may be associated with a damaged or dysfunctional epithelium. Also, changes in the activities of protein kinase C have been implicated in the pathogenesis of asthma. This study examined the role of protein kinase C in the modulation of airway smooth muscle tone and the influence of the epithelium on this function. Phorbol-12,13-diacetate (PDA) (10(-8) to 10(-5) M) induced concentration-dependent and epithelium-independent relaxations of guinea pig tracheal rings. PDA (10(-8) to 10(-5) M) induced significantly greater relaxations of tracheal rings contracted with 5-hydroxytryptamine (10(-5) M) than in tissues contracted to an equivalent degree with acetylcholine (10(-6) M). In experiments using phenoxybenzamine (10(-7) M and 10(-5) M), the dissociation constant (KA) for acetylcholine was significantly greater than that for 5-hydroxytryptamine. The fraction of active receptors (q) calculated for acetylcholine was significantly smaller than that calculated for an equieffective concentration of 5-hydroxytryptamine. Relaxations to PDA in tissues contracted with acetylcholine (2 x 10(-6) M) or 5-hydroxytryptamine (10(-5) M) were significantly augmented by phenoxybenzamine (10(-5) M and 10(-7) M, respectively). PDA did not affect contractions to acetylcholine (10(-8) to 10(-3) M) in the presence of epithelium but caused a significant right-ward displacement of the acetylcholine concentration-contraction curve in the absence of epithelium. The concentration-contraction curves for 5-hydroxytryptamine (10(-8) to 10(-5) M) were significantly displaced to the right by PDA in the presence or absence of epithelium. This effect was greater in the absence of epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)     

The contractile response of the mesenteric resistance arteries to prostaglandin F2alpha; effects of simultaneous hyperlipemia-diabetes

The effect of hyperlipemia associated with diabetes on the contractility of resistance arteries to prostaglandin F2alpha (PGF2alpha) was investigated employing 4 weeks simultaneously hyperlipemic-diabetic (HD), hyperlipemic (H), diabetic (D) and normal hamsters (controls, C). The isometric force produced by explanted arteries in the presence of 10(-8) to 10(-5) M PGF2alpha was recorded by the myograph technique. The results showed that compared with controls, the contractile response to 10(-5) M PGF2alpha was approx. 2 fold increased in HD group, and approx. 1.75 and 1.62-fold enhanced in H and D groups, respectively. Activation of protein kinase C with 10(-6) M phorbol 12-myristate 13-acetate increased the contractility to PGF2alpha in all groups and particularly in HD hamsters (approx. 10.16-fold). Inhibition of cyclooxygenase by indomethacin increased approx. 1.81-fold the arterial contractility to PGF2alpha in C group, whereas in H, D and HD hamsters had no effect. Blockage of Ca(2+)-activated K(+)-channels with 10(-3) M tetraethylammonium augmented the contraction to PGF2alpha approx. 6.43-fold in C group, and at significantly lower levels in H, D and HD groups, i.e. approx. 3.84, 3.72 and 3.33-fold, respectively. The results validate two conclusions: (i) simultaneous insult of hyperlipemia-hyperglycemia is associated with the highest contractility of the resistance arteries to PGF2alpha; the highest circulating glucose and cholesterol levels, and the enhancement in the protein kinase C pathway underlay the augmented contractility; (ii) no matter the pathology induced (hyperlipemia, diabetes or both simultaneously) a common dysfunctional response to PGF2alpha was installed; this consists in a reduced effect of cyclooxygenase inhibition, and a altered activity of Ca(2+) dependent K(+) channels.     

Inhibition by lithium of the hydroosmotic action of vasopressin in the isolated perfused cortical collecting tubule of the rabbit

Because treatment with lithium salts may impair renal concentrating ability, we investigated the possibility of a direct effect of lithium ions on the permeability to water of the collecting duct epithelium. The coefficient of hydraulic conductivity (Lp) of isolated perfused rabbit cortical collecting tubules (CCT) was measured in the presence and absence of arginine-8-vasopressin (AVP), or 8-bromo (Br) cyclic AMP (cAMP) and/or lithium chloride (Li 10 mM). In the absence of AVP, Li in the lumen for 30 min failed to affect basal water permeability; however, in tubules preincubated with Li in the lumen for 80 min, basal water permeability was reduced to 30% of the value found in control tubules (P less than 0.01). In CCT incubated at 25 degrees C with Li in the lumen for 3 h, the hydroosmotic response to 2.5 microU X ml-1 AVP (Lp = 6.88 +/- 1.54 nl X cm-2 X s-1 X atm-1) was significantly lower than that in the control tubules (13.98 +/- 1.59, P less than 0.01); the inhibition was not reversible. When Li was present in the peritubular medium only, the hydroosmotic effect of AVP was not different from that of the controls. The hydroosmotic effect of 25 microU/ml AVP was investigated at 37 degrees C. CCT exposed to Li in the lumen had a 49% inhibition of peak Lp under AVP (Lp = 10.98 +/- 1.17) as compared with control tubules (Lp = 21.39 +/- 1.51; P less than 0.005). In contrast, the hydroosmotic response to 8-Br-cAMP was not affected by lithium. The results are compatible with the view that Li inhibits the action of AVP at the level of the regulating protein or the catalytic unit of the membrane adenylate cyclase and that the site of the interaction can be reached by lithium only from the cytoplasmic side. The Li-antidiuretic hormone (ADH) interaction found here may represent the earliest pathophysiological event underlying the renal concentrating defect observed after Li administration.     

Endothelin's biphasic effect on fluid absorption in the proximal straight tubule and its inhibitory cascade.

The effect of endothelin-1 (ET-1) on the proximal tubule remains unclear. This may be due to a biphasic effect on transport in this segment. We hypothesized that ET-1 has a biphasic effect on fluid absorption (Jv) in the proximal straight tubule and that its inhibitory effect is superimposed on its stimulatory effect. ET-1 (10(-13) M) stimulated Jv from 0.68 +/- 0.07 to 1.11 +/- 0.20 nl/mm/min, a 60% increase (P < 0.04). 10(-12) and 10(-10) M ET-1 had no significant effect. 10(-9) M ET-1 reduced Jv from 0.81 +/- 0.19 to 0.44 +/- 0.15 nl/mm/min (P < 0.009). Staurosporine (STP, 10(-8) M) prevented both 10(-9) and 10(-13) M ET-1 from altering Jv significantly indicating that protein kinase C (PKC) is involved. Indomethacin (10(-5) M) blocked the inhibition produced by 10(-9) M ET-1. ETI (10(-6) M), a lipoxygenase inhibitor, also blocked ET-1 inhibition of Jv. Interestingly ET-1 (10(-9) M) stimulated Jv in the presence of both indomethacin and ETI. When 10(-9) M ET-1 was added in the presence of 10(-5) M quinacrine, a phospholipase (PL) inhibitor, Jv also increased from 1.02 +/- 0.20 to 1.23 +/- 0.22 nl/mm/min (P < 0.03). STP blocked this increase. We conclude that (a) 10(-13) M ET-1 stimulates fluid absorption by activating PKC; (b) 10(-9) M ET-1 decreases Jv by PKC-, PL-, cyclooxygenase-, and lipoxygenase-dependent mechanisms; and (c) the inhibitory effect of ET-1 on Jv is superimposed on the stimulatory effect.     

Insulin-like growth factor I gene expression in isolated rat renal collecting duct is stimulated by epidermal growth factor.

The renal collecting duct is a site of insulin-like growth factor I (IGF I) synthesis. Epidermal growth factor (EGF) is also synthesized within the kidney in the thick ascending limb of Henle's loop and the distal tubule. EGF has been shown to regulate IGF I expression in nonrenal tissues. To shed light upon a role of EGF in intrarenal regulation of IGF I gene expression, plasma membranes prepared from collecting ducts isolated from rat kidney and collecting ducts themselves were incubated in the presence and absence of recombinant human EGF (hEGF). hEGF enhanced phospholipase C activity in collecting duct plasma membranes establishing the potential for EGF signal transduction at this site. Inclusion of hEGF in suspensions of collecting ducts increased production of immunoreactive IGF I in a concentration-dependent manner. Production was stimulated significantly by addition of 10(-8) or 10(-6) M hEGF to suspensions for 2 h. Levels of IGF I mRNA in collecting ducts were increased 2.8-fold after incubation with 10(-6) M hEGF in vitro. Our findings demonstrate a direct action of hEGF to enhance collecting duct IGF I gene expression in vitro. Such enhancement is likely to reflect an effect of EGF to stimulate IGF I production in the collecting duct of the intact kidney. Since EGF is produced in kidney, our findings are consistent with intrarenal paracrine regulation of IGF I gene expression by EGF.     

Beneficial effects of calcium channel blockers and calmodulin binding drugs on in vitro renal cell anoxia

Proximal tubules (PSTs) of the S1, S2 and S3 segments and cortical collecting tubules (CCTs) were microdissected individually from rabbit kidneys and cultured for 7 days in hormonally defined media. Anoxia was induced by incubation of cultures in normal medium for 45 min at 25 degrees C in an atmosphere of nitrogen and cell death was measured by nigrosine dye uptake. After 45 min of anoxia and a 4- to 6-hr incubation in normal Ca++-containing media, cells from all segments were dead. Addition of calcium channel blockers verapamil and nifedipine (5 X 10(-7) and 10(-6) M, respectively) for the first 2 hr after anoxia to the incubation media was associated with a 60 +/- 8 and 33 +/- 7% survival of PST cells (5 hr after anoxia), p less than .05. Verapamil at 5 X 10(-8) M caused a 42 +/- 4% survival whereas nifedipine at 10(-7) M was not effective on the survival rate of PST cells (5 hr after anoxia). These calcium channel blockers also afforded protection from anoxic cell death for CCT cells. The role of calmodulin in anoxic cell injury was studied by means of calmodulin binding drugs, trifluoperazine and W7 [N-(6-aminohexyl)-5-chloronapthalene-sulfonamide]. Addition of trifluoperazine (5 X 10(-7) M) and W7 (5 X 10(-7) M to both PST and CCT cells during the 2-hr reflow period after 45 min of anoxia increased viability by 58 +/- 3 and 62 +/- 3%, respectively (P less than .05) at 5 hr postanoxia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gentamicin inhibits agonist stimulation of the phosphatidylinositol cascade in primary cultures of rabbit proximal tubular cells and in rat renal cortex

A growing body of evidence indicates that aminoglycoside antibiotics interact with phosphoinositides and this has led to the hypothesis that these drugs perturb the phosphatidylinositol (PI) cascade. To test this hypothesis we examined the effect of gentamicin on agonist stimulation of the PI cascade in primary culture of rabbit proximal tubular cells (RPTC) and in rat renal cortex. Parathyroid (PTH) (10(-6) M) stimulated a significant increase in total inositol phosphates, inositol monophosphate and inositol trisphosphate, but not inositol bisphosphate in RPTC with the peak effect at 2 min. This effect was completely inhibited in RPTC exposed to 10(-3) M gentamicin for 48 and 24 hr. In other experiments we demonstrated that angiotensin II, phenylephrine, bradykinin and arginine vasopressin (all at 10(-6) M) stimulated inositol trisphosphate generation in control RPTC but not in cells exposed to 10(-3) M gentamicin for 24 h. In contrast gentamicin did not block PTH-stimulation of cyclic AMP generation, which indicates that gentamicin did not prevent PTH from interacting with its plasma membrane receptor. PTH also stimulated redistribution of protein kinase C from the cytosolic to the membrane fraction of RPTC. This effect was completely abolished in RPTC exposed to 10(-3) M gentamicin for 2 days. PTH given i.p. to rats stimulated the redistribution of protein kinase C from the cytosolic to the membrane fraction of renal cortex. This effect was completely inhibited in rats injected with gentamicin, 100 mg/kg per day for 2 days. The     

Effects of protein kinase inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, on beta-2 adrenergic receptor activation and desensitization in intact human lymphocytes

To investigate the role of protein kinases in agonist-mediated beta-2 adrenergic receptor regulation, the effects of the protein kinase A and C inhibitor, H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], on isoproterenol-induced beta adrenoceptor activation and desensitization have been studied in intact human lymphocytes. In the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, H-7 potentiated 10(-8) to 10(-4) M isoproterenol or prostaglandin E1-induced cyclic AMP (cAMP) accumulation in a dose-dependent manner. We failed to observe any effect of H-7 on forskolin-induced cAMP accumulation. These effects of H-7 are probably not due to its inhibition of phosphodiesterase. In addition, whereas up to 10(-3) M H-7 had no beta adrenergic receptor blocking effect, preincubation of intact cells with 10(-3.5) M H-7 partially prevented 50 nM isoproterenol-induced beta-2 adrenergic receptor desensitization in terms of decreases in beta adrenoceptor density (maximum binding), isoproterenol-mediated cAMP responsiveness and high affinity receptor binding for agonist. Interestingly, 10(-3.5) M H-7 alone treated cells also showed an up-regulation of cell surface beta receptor density (maximum binding) and increased cAMP responsiveness to isoproterenol stimulation. The mechanisms are unclear. If these effects occur as a result of inhibition by H-7 of protein kinase A and/or C, it may suggest an important role of protein kinase A and/or C in agonist-induced beta-2 adrenergic receptor regulation.     

Cyclic adenosine monophosphate and diacylglycerol. Mutually inhibitory second messengers in cultured rat inner medullary collecting duct cells.

Studies were performed to examine interactions between the adenylyl cyclase (AC) and phospholipase C (PLC) signaling systems in cultured rat inner medullary collecting duct cells. Stimulation of AC by either arginine vasopressin (AVP) or forskolin or addition of exogenous cAMP inhibits epidermal growth factor (EGF)-stimulated PLC. This inhibition is mediated by activation of cAMP-dependent kinase as it is prevented by pretreatment with the A-kinase inhibitor, N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide (H8) but not by the C-kinase inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7). Exposure to EGF eliminates AVP-stimulated cAMP generation. This is not mediated by a cyclooxygenase product as inhibition by EGF is observed even in the presence of the cyclooxygenase inhibitor, flurbiprofen. Inhibition by EGF is not due to an increase in inositol trisphosphate (IP3) as exposure of saponin-permeabilized cells to exogenous IP3 is without effect. Inhibition by EGF is prevented by pretreatment with the C-kinase inhibitor, H7, but not by the A-kinase inhibitor, H8. Exposure to the synthetic diacylglycerol (DAG), dioctanoylglycerol, also inhibits AVP-stimulated AC activity; therefore, inhibition by EGF is due to activation of protein kinase C. Thus, in cultured rat inner medullary collecting duct cells, cAMP and DAG function as mutually inhibitory second messengers with each impairing formation of the other.     

Bradykinin B2 receptor activates extracellular signal-regulated protein kinase in mIMCD-3 cells via epidermal growth factor receptor transactivation

Bradykinin (BK) has been implicated in the regulation of renal function. Activation of extracellular signal-regulated protein kinase (ERK1/2) has been demonstrated in several models of toxic or proliferative renal injury. We studied activation of ERK1/2 by BK in a cell model of the most distal part of the nephron, inner medullary collecting duct (mIMCD-3) cells. Exposure of mIMCD-3 cells to BK (10(-10)-10(-5) M) resulted in a concentration-dependent increase in tyrosine phosphorylation of ERK1/2, with maximal effect at 10(-8) M BK. ERK1/2 activation by BK was observed as early as 1 min, peaked at 5 min, and was sustained at least for 1 h. The effect of BK was mediated by the B(2) receptor and was pertussis toxin-independent. Inhibition of phospholipase C, protein kinase C, or phosphatidylinositol 3-kinase did not alter ERK1/2 activation by BK. BK-induced ERK1/2 activation was Ca(2+)-calmodulin-independent but was sensitive to genistein, an inhibitor of tyrosine kinase(s). AG1478, a specific inhibitor of epidermal growth factor receptor (EGFR) kinase, completely blocked the effect of BK, suggesting an essential role of EGFR in ERK1/2 activation by BK. Immunoprecipitation/Western blot studies revealed that BK stimulated tyrosine phosphorylation of EGFR, its association with an adapter molecule Grb2, and complex formation between Grb2 and the adapter protein Shc. Activation studies of monomeric G protein Ras showed that BK-induced stimulation of Ras was dependent on EGFR tyrosine kinase activity. These studies demonstrate that BK stimulates Ras-dependent activation of ERK1/2 in mIMCD-3 cells via transactivation of EGFR through a novel mechanism.