Epidermal growth factor-stimulated phosphoinositide hydrolysis in cultured rat inner medullary collecting tubule cells. Regulation by G protein, calcium, and protein kinase C.

Epidermal growth factor (EGF) exhibits specific saturable binding to cultured rat inner medullary collecting tubule cells and stimulates inositol trisphosphate (IP3) production by these cells in a dose-dependent fashion. EGF-stimulated IP3 production is enhanced by GTP gamma s or AIF4- and is inhibited by GDP beta s or pertussis toxin. Alterations in extracellular Ca2+ have no effect on either basal or EGF-stimulated IP3 production. Similarly, treatment with EGTA which decreases cytosolic Ca2+ is without effect. In contrast, treatment with ionomycin which increases cytosolic Ca2+ has no effect on basal IP3 production but enhances the response to EGF. Activation of protein kinase C inhibits IP3 production in response to either EGF or AIF4-. These studies demonstrate the occurrence of EGF-stimulated phospholipase C activity in the rat inner medullary collecting duct. Stimulation by EGF is transduced by a pertussis toxin-sensitive G protein, unaffected by alterations in extracellular Ca2+, insensitive to a decrement in cytosolic Ca2+, enhanced by an increase in cytosolic Ca2+, and inhibited by protein kinase C.

AVP stimulates adenylyl cyclase and phospholipase C in reciprocal fashion in cultured RIMCT cells.

In cultured rat inner medullary collecting tubule (RIMCT) cells, arginine vasopressin (AVP) stimulates adenylyl cyclase (AC) activity in dose-dependent fashion, with no response at concentrations of 10(-10) M or below and with peak activity at 10(-7) M AVP. In contrast, AVP-stimulated phospholipase (PLC) activity is greatest at concentrations at which there is no effect on AC and decreases at higher concentrations of AVP, becoming undetectable at 10(-7) M. Increasing cellular adenosine 3',5'-cyclic monophosphate (cAMP) content with either exogenous ClPheScAMP or forskolin eliminates inositol trisphosphate production in response to 10(-13) M AVP. Conversely, inhibition of AC by 2',5'-dideoxyadenosine (DDA) unmasks PLC activity in response to 10(-7) M AVP that is not observed in the absence of DDA. Similarly, DDA prevents inhibition of epidermal growth factor-stimulated PLC by AVP. These findings demonstrate the reciprocal relationship between AVP-stimulated AC and PLC activities in cultured RIMCT cells, which may explain previous divergent results regarding the ability of AVP to stimulate PLC in this tissue.

Adrenergic control of cAMP generation in rat inner medullary collecting tubule cells.

The adrenergic nervous system profoundly alters water excretion by both renal and extrarenal pathways. The effects of catecholamines on cultured rat inner medullary collecting tubule cells were studied. The beta-adrenergic agonist, isoproterenol, increases cAMP from 19.5 +/- 2.3 to 79.4 +/- 14.4 fm/micrograms protein, P less than 0.001. The response to arginine vasopressin (AVP) is also greater in the presence of isoproterenol, but the increment is unchanged when compared to that seen in the absence of AVP. The agonist effect of isoproterenol is blocked by propranolol but not by the specific beta 1 antagonist, atenolol. The effect of alpha-adrenergic stimulation was studied by the use of norepinephrine (NE) in the background of the beta blocker, propranolol. NE decreases AVP-stimulated cAMP generation from 190 +/- 11 to 117 +/- 10 fm/micrograms, P less than 0.001, N = 6. The specific alpha 2 antagonist, yohimbine, but not the alpha 1 antagonist, prazosin, prevents the NE-induced decrease as AVP-stimulated cAMP is restored to 187 +/- 19 fm/micrograms. Similarly the selective alpha 2 agonist, clonidine, significantly inhibits both AVP- and isoproterenol-mediated cAMP generation. To define the site of alpha 2 inhibition in the adenylate cyclase (AC) complex the effect of pertussis toxin (PT) was investigated. After pretreatment with PT (1-1000 ng/ml), AVP-stimulated cAMP was not inhibited by NE. The alpha 1 agonist, phenylephrine, fails to inhibit AC or to increase cytosolic Ca in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal regulation of MAP kinase in cultured rat inner medullary collecting tubule cells.

Mitogen-activated protein (MAP) kinase is a widely expressed protein serine/threonine kinase that serves as a convergence point for many signaling pathways including receptor tyrosine kinases, G protein-coupled receptors, and protein kinase C (PKC). The hormonal regulation of MAP kinase was studied in cultured established rat inner medullary collecting tubule (RIMCT) cells. Neither vasopressin nor beta-adrenergic agonists stimulated MAP kinase, despite clear stimulation of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. In contrast, carbachol, ATP, and epidermal growth factor (EGF), which are known to antagonize vasopressin action in the RIMCT, stimulated the MAP kinase pathway. This stimulation was mimicked by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, which directly activates PKC. The potency with which EGF and carbachol activated MAP kinase was similar to the potency with which they inhibited vasopressin-stimulated cAMP accumulation. To assess the role of Gi proteins in these stimulatory events, RIMCT cells were pretreated with pertussis toxin to inhibit Gi-mediated signaling. Pertussis toxin did not influence ATP- or EGF-stimulated MAP kinase, but completely inhibited carbachol stimulation, suggesting that Gi proteins mediate muscarinic stimulation. Prolonged exposure of RIMCT cells to high phorbol ester concentrations to downregulate PKC ablated carbachol- and ATP-stimulated MAP kinase, but not EGF-stimulated MAP kinase, suggesting that PKC is a component of the network involved in MAP kinase activation by purinergic and muscarinic agonists. Investigation of the sidedness of the hormonal stimulations indicated that EGF-stimulated MAP kinase was highly polarized, occurring exclusively from the basolateral surface, whereas carbachol stimulated MAP kinase similarly from either cell surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)