New signal transduction mechanisms of atrial natriuretic factor: inhibition of phosphorylation of protein kinase C and A 240 kDa protein in adrenal cortical plasma membrane by cGMP dependent and independent mechanisms

The effects of synthetic atrial natriuretic factor (ANF) on the state of protein phosphorylation in plasma membranes of bovine adrenal cortex have been studied in vitro. ANF (1x10(-8)M - 1x10(-7)M) specifically inhibited the phosphorylation of two distinct proteins of 78 kDa and 240 kDa. Immunoblotting with specific antiserum to protein kinase C produced evidence that 78 kDa protein is most likely the protein kinase C whose phosphorylation is inhibited by both ANF and cGMP. However, cGMP did not affect the phosphorylation of 240 kDa protein, indicating a new cGMP-independent mechanism of ANF action in the adrenal, which is compatible with the lack of action of cGMP and its analogs in ANF-induced inhibition of aldosterone secretion from adrenal cortex. The inhibition of phosphorylation of putative protein kinase C by ANF or cGMP indicates a hitherto unknown signal transduction mechanism of ANF.     

Atrial natriuretic factor stimulates the frog urinary bladder osmotic permeability in presence of a cyclic nucleotide phosphodiesterase inhibitor

The role of atrial natriuretic factor (ANF) in regulation of osmotic water permeability was studied in isolated frog Rana temporaria L. urinary bladder. It was found that ANF (rANF, 1-28) added to the serosal solution at concentrations 5 x 10(-8) M and higher dosedependently stimulated the arginine-vasotocin (AVT)-induced increase of osmotic water permeability. The effect of ANF was revealed only in presence of 3-isobuthyl-1-methylxantine (180 microM) and was accompanied by significant elevation of cGMP level in urinary bladder homogenate and isolated mucosal epithelial cells. C-ANF (des[Gln18, Ser19, Gly20, Leu21, Gly22]-ANF-(4-23)-NH2), a specific agonist of NPR-C receptor, exerted no effect on osmotic water permeability. ANF induced a significant increase of cAMP in urinary bladder homogenates (AVT, 5 x 10(-11) M: 52.3 +/- 10.6; AVT + ANF, 10(-7) M: 114.2 +/- 26.9 pmol/mg protein, n = 5, p < 0.05). The activity of adenylate cyclase in crude plasmatic membrane fraction was not changed. Milrinone, a specific inhibitor of phosphodiesterase 3, at concentrations from 25 to 80 microM, enhanced both the hydroosmotic response to AVT and AVT-stimulated cAMP production. Altogether these data demonstrate that, in the frog urinary bladder, ANF stimulates the AVT-induced increase of osmotic water permeability acting probably through NPR-A receptor-coupled mobilization of cGMP and cGMP-dependent inhibition of phosphodiesterase 3.     

Identification and characterization of three distinct atrial natriuretic factor receptors. Evidence for tissue-specific heterogeneity of receptor subtypes in vascular smooth muscle, kidney tubular epithelium, and Leydig tumor cells by ligand binding, photoaffinity labeling, and tryptic proteolysis

Three distinct atrial natriuretic factor (ANF) receptors have been identified and characterized from rat thoracic aortic cultured vascular smooth muscle (RTASM) cells, kidney tubular epithelium (MDCK), and Leydig tumor (MA-10) cells. These include 1) a disulfide-linked 140-kDa protein found in RTASM cells, which was reduced by dithiothreitol (DTT) to 70 kDa, 2) a 120-135-kDa single polypeptide protein, specific to MDCK and MA-10 cells whose Mr was not reduced by DTT, and 3) a 66-70-kDa protein prevalent in both RTASM and MDCK cells, which was not reduced by DTT. After incubation of RTASM cells with 4-azidobenzoyl 125I-ANF, labeling of the 140-kDa protein was blocked by both full-length ANF(99-126) and truncated ANF103-123. In contrast, the labeling of the 120-kDa receptor in MDCK cells was blocked only by full-length ANF(99-126). However, labeling of the 68-70-kDa receptor in both RTASM and MDCK cells was blocked by full-length ANF(99-126) and truncated ANF(103-123). Binding of 125I-ANF(99-126) to RTASM and MDCK cells was rapid, specific, and saturable with a Kd of 1.5 x 10(-10) M and binding capacity (Bmax) of 2.1 x 10(5) sites/RTASM cell and Kd 4.5 x 10(-10) M and Bmax 5 x 10(4) sites/MDCK cell, respectively. Binding of 125I-ANF(99-126) to RTASM cells was displaced with both full-length ANF(99-126) and truncated ANF(103-123), however, binding to MDCK cells was efficiently displaced only with full-length ANF. Both ANF(99-126) and ANF(103-123) stimulated cGMP in RTASM cells but only ANF(99-126) elicited cGMP in MDCK cells. Tryptic proteolysis of the high Mr single chain receptor produced only a 68-kDa fragment, whereas disulfide-linked 140-kDa receptor yielded 52-, 38-, 26-, and 14-kDa fragments. These data provide direct biochemical evidence for three distinct ANF receptors which might be linked to diverse physiological functions of ANF such as natriuresis in the kidney, vasorelaxation in vascular smooth muscle, and steroidogenic responsiveness in Leydig cells.     

Atrial natriuretic factor inhibits phosphate uptake in opossum kidney cells: as a model of renal proximal tubules

The cultured renal cell, an opossum kidney (OK) cell line, which contains several features characteristic of proximal tubular cells, was utilized to examine the direct effects of atrial natriuretic factor (ANF) and cyclic GMP (cGMP) on phosphate uptake. ANF at 2 x 10(-7) M significantly inhibited phosphate uptake by 10.1% of control (P less than 0.01). Incubation of the cells with ANF (10(-8) to 10(-6) M) resulted in an increment of intracellular cGMP in a dose dependent fashion. Exogenous addition of 8-bromo-cGMP (10(-4) M) also significantly inhibited phosphate uptake by 14.6%. These results suggest that ANF directly inhibits phosphate transport in renal proximal tubular cells, probably through stimulation of cGMP production.     

Principal neurofilament-associated protein kinase in squid axoplasm is related to casein kinase I

A cytoskeletal extract of pure axoplasm, highly enriched with neurofilaments (ANF), was prepared from the giant axon of the squid. This ANF preparation also contained potent kinase activities which phosphorylated the Mr greater than 400,000 (high molecular weight) and Mr 220,000 squid neurofilament protein subunits. High salt (1 M) extraction of this ANF preparation solubilized most of the neurofilament proteins and kinase activities and gel filtration on an AcA 44 column separated these two components. The neurofilaments eluted in the void volume of the column while the kinase activities eluted in the 17-44-kDa range of the column. Two major kinase activities were measured in this peak of activity. One of these strongly phosphorylated the phosphate acceptor peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) and was completely inhibited by the selective inhibitor of cAMP-dependent kinase Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile- NH2 (Wiptide). Since addition of cAMP did not stimulate activity, this suggested that this kinase was a free catalytic subunit of cAMP-dependent kinase associated with the neurofilaments. The second kinase activity most effectively phosphorylated alpha-casein, and this activity was not affected by Wiptide. The alpha-casein phosphorylating activity (ANF kinase) was the principal activity responsible for neurofilament protein phosphorylation, and was not inhibited by various inhibitors against second messenger regulated kinases, suggesting it was related to the casein kinase family. Four lines of evidence indicate ANF kinase was similar to casein kinase I. These were: 1) the apparent molecular weight determined by gel filtration and the chromatographic elution profile on phosphocellulose column corresponded to casein kinase I; 2) heparin, an inhibitor of casein kinase II at 2-5 micrograms/ml, stimulated both ANF kinase and purified casein kinase I at these concentrations, while CKI-7, a relatively selective inhibitor of casein kinase I, inhibited ANF kinase in a comparable dose-response fashion; 3) purified casein kinase I strongly phosphorylated both ANF protein subunits (like ANF kinase) whereas casein kinase II was relatively ineffective; and 4) tryptic peptide maps of the HMW and Mr 220,000 neurofilament proteins after phosphorylation by ANF kinase or purified casein kinase I showed similar 32P-peptide patterns.     

Cyclic AMP-dependent phosphorylation of filamin in mammalian smooth muscle.

Filamin is a high molecular weight actin-binding protein found in large quantities in smooth muscle and other non-muscle cells. We have studied the phosphorylation of filamin in a mammalian smooth muscle, the guinea pig vas deferens. Intact vas deferens incorporated [32P]orthophosphate into filamin. Incubation of particulate fractions of vas deferens with [gamma-32P]ATP resulted in 32P-labeling of filamin. Cyclic AMP stimulated this phosphorylation, whereas cyclic GMP and Ca2+ had no effect. Purified vas deferens filamin can be phosphorylated by purified cyclic AMP-dependent protein kinase. We have compared cyclic AMP and cyclic GMP effects on phosphorylation in smooth muscle. Cyclic GMP stimulated phosphorylation of two particulate proteins, G-I (Mr = 130,000) a protein previously described by Casnellie, J. E., and Greengard, P. (1974) Proc. Natl. Acad, Sci. U.S.A. 71, 1891-1895 and G-III (Mr = 240,000). Both proteins and the kinase responsible for their phosphorylation appear to be membrane-bound. Phosphorylation of both proteins is stimulated by cyclic GMP (Ka = 3 x 10(-8) M), cyclic AMP (Ka = 3 x 10(-7) M), and to a lesser degree by Ca2+. In contrast, filamin phosphorylation is due to a soluble kinase stimulated only by cyclic AMP (Ka = 3 x 10(-7) M) and not by cyclic GMP or Ca2+.     

Endothelin inhibits the atrial natriuretic factor stimulated cGMP production by activating the protein kinase C in rat aortic smooth muscle cells.

Preincubation of rat thoracic aortic smooth muscle cells with endothelin inhibits the atrial natriuretic factor (ANF)-induced cGMP accumulation in these cells in a concentration dependent manner. The maximal inhibition of 64% was afforded by 1 x 10(-6) M endothelin and the half maximal inhibition (IC50) was achieved with 1 x 10(-9) M endothelin. Endothelin (1 x 10(-6) M) also increased the plasma membrane bound protein kinase C (PKC) activity by 4 fold. Hormone-dependent increase in PKC activity was limited to plasma membranes only and some decrease in cytosolic PKC activity was observed. However, phorbol 12-myristate 13-acetate (PMA) (1 x 10(-6)M) provoked a total loss of cytosolic PKC activity and a net gain in membranous PKC activity indicative of the translocation of the enzyme. Pretreatment of these cells with H-7, a PKC inhibitor, released the endothelin and PMA-mediated attenuation of ANF-stimulated cGMP formation. These results suggest that PKC is involved in the regulation of ANF-induced cGMP accumulation and that the vasoconstrictor activity of endothelin might involve inhibition of the vasorelaxant activity of ANF through the inhibition of cGMP accumulation in smooth muscle cells (SMCs) of the rat aorta.     

Phorbol and calcium decreased atriopeptin response in a human renal cell line.

We investigated regulation of atrial natriuretic factor (ANF)-stimulated cellular cGMP accumulation (ANF-s-cGMP) in an ANF-responsive human renal cell line, SK-NEP-1. Dose-response data indicated that the EC50 for ANF(99-126) was 1.1 x 10(-9) M. Brain natriuretic peptide (10(-6) M) increased cGMP to a level indistinguishable from that of ANF (10(-6) M). [Met-(O)]ANF was only half as potent as ANF, and atriopeptin I (10(-6) M) did not increase cGMP over basal levels. Preincubation of SK-NEP-1 cells with ANF, but not atriopeptin I (API), for two hours or longer, caused a concentration-dependent down-regulation of ANF-s-cGMP. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, and A23187 and its 4-bromo derivative, calcium ionophores, inhibited ANF-s-cGMP in a dose-dependent manner. A23187 inhibition was calcium dependent and promoted net cGMP degradation. Thirty-six hour preincubation with PMA, a procedure used to down-regulate PKC, abolished acute PMA inhibition of ANF-s-cGMP without having an effect on ANF-s-cGMP or on 4-bromo-A23187 inhibition thereof. These data indicate that PKC activation specifically inhibited ANF-s-cGMP but that PKC was not required for ANF-s-cGMP in SK-NEP-1 cells. Thus structurally related ANF peptides, protein kinase C (PKC) activators, calcium ionophores are potential modulators of ANF-s-cGMP in cells from this human renal cell line.     

Atrial natriuretic factor-induced cGMP accumulation in rat anterior pituitary cells in culture is not coupled to hormonal secretion

While atrial natriuretic factor (ANF) does not influence ACTH secretion, it was reported to have a marked stimulatory effect on the intracellular accumulation of cGMP in rat anterior pituitary cells in culture. Since many biological actions of ANF appear coupled to its excitatory action on target cell guanylate cyclase, the current study was designed to characterize the ANF-induced cGMP response in anterior pituitary with a view to determining whether the nucleotide plays a regulatory role in the secretory function of this gland. A 3 min exposure of cells in primary culture to 300 nM ANF (99-126) or 100 microM sodium nitroprusside (SNP), a stimulator of guanylate cyclase, causes maximal 10- and 3-fold elevations of cGMP levels, respectively. Following a progressive decrease, 6- and 2-fold increases over basal cGMP levels are still observed after 180 min of incubation with ANF (99-126) and SNP, respectively. The half-maximal stimulation of cGMP accumulation induced by a 10 min exposure to ANF (99-126), or rat atriopeptin II (ANF 103-125) is observed at 9 +/- 2 and 125 +/- 22 nM, respectively. ANF fragments (99-109) and (111-126), as well as human cardiodilatin (hANF 1-16), do not alter cGMP levels. Basal and ANF-induced cGMP levels are at least 10-fold higher in cell populations enriched in gonadotrophs compared to gonadotroph-impoverished preparations. A 3 h incubation of cells with ANF (0.1-1000 nM), however, fails to modify spontaneous or LHRH-induced LH secretion. Similarly, ANF does not alter spontaneous release of GH, TSH or PRL. The data suggest indirectly that gonadotrophs represent a principal site at which ANF acts to stimulate cGMP synthesis, but that the nucleotide is not a specific regulator of the LH secretory process; nor is it generally involved as a second messenger in the secretory function of any cell type of the anterior pituitary gland.     

Cyclic 3',5'-adenosine monophosphate-dependent kinase and phosphoproteins in human amnion

3',5'-Cyclic adenosine monophosphate (cAMP) modulates prostaglandin production in human amnion membranes. The major effects of cAMP are presumably mediated through the phosphorylation of specific regulatory phosphoproteins following cAMP activation of cAMP-dependent protein kinase. Cyclic AMP-dependent protein kinase and phosphoproteins have not previously been characterized in human amnion. Total homogenates, cytosol, and membrane fractions from human amnion were examined for [3H]cAMP binding activity and cAMP-dependent kinase activity. cAMP-dependent kinase activity was barely detectable in crude amnion fractions. Cytosol was therefore partially purified by DEAE column chromatography for further examination. Two peaks of coincident [3H]cAMP binding and cAMP-dependent kinase activity were demonstrated at 70 and 140 mM NaCl, characteristic of the Type I and Type II cAMP-dependent protein kinase isozymes. [3H]cAMP binding to the material from both peak fractions was saturable and reversible. Scatchard analysis of [3H]cAMP binding to the peak fractions was linear for peak I and curvilinear for peak II. Assuming a one-site model, [3H]cAMP binding to the Type I isozyme showed a KD = 4.17 x 10(-8) M and Bmax = 73 pmole/mg protein; using a two-site model, [3H]cAMP binding to the high-affinity site for the Type II isozyme had a KD = 3.94 x 10(-8) M and Bmax = 6.3 pmole/mg protein. Other cyclic nucleotides competed for these [3H]cAMP binding sites with a potency order of cAMP much greater than cGMP greater than (BU)2cAMP.cAMP caused a dose-dependent increase in cAMP-dependent kinase activity in the peak fractions; half-maximal activation was observed with 5.0 x 10(-8) M cAMP. The ability of cAMP to increase phosphorylation of endogenous proteins in both crude amnion cytosol and cytosol from cultures of amnion epithelial cells was assessed using [32P]ATP, SDS-polyacrylamide gel electrophoresis and autoradiography. cAMP stimulated 32P incorporation into three proteins having Mr = 80,000, 54,000, and 43,000 (P less than .01). Half-maximal 32P incorporation into these proteins occurred at 1.0 x 10(-7) M cAMP. cAMP-dependent kinase is present in human amnion; specific cAMP-enhanced phosphoproteins are also present. Hormones elevating cAMP levels in amnion may exert their effects by activating cAMP-dependent kinase and phosphorylating these phosphoproteins.     

Physiological Ca2+ level and Ca2+-induced Permeability Transition Pore control protein phosphorylation in rat brain mitochondria

Phosphorylation of several low molecular mass proteins (3.5, 17, 23 and 29kDa) was observed in rat brain mitochondria (RBM) at ATP concentration close to that in the mitochondrial matrix. Furthermore, regulatory effects of Ca2+ on phosphorylation of these proteins were investigated. Protein phosphorylation was found to be modulated by Ca2+ in the physiological concentration range (10(-8) to 10(-6)M free Ca2+). Incorporation of 32P from [gamma-32P]ATP into the 17kDa protein was dramatically increased within the 10(-7) to 10(-6)M free Ca2+ range, whereas an opposite effect was observed for the 3.5kDa polypeptide. Strong de-phosphorylation of the 3.5kDa polypeptide and enhanced 32P-incorporation into the 17 and 23kDa proteins were found with supra-threshold Ca2+ loads and these effects were eliminated or reduced in the presence of cyclosporin A, an inhibitor of Permeability Transition Pore (PTP) opening. In the presence of calmidazolium (Cmz), a calmodulin antagonist, enhanced levels of phosphorylation of the 17 and 3.5kDa polypeptides were observed and the 17kDa protein phosphorylation was suppressed by H-8, a protein kinase A inhibitor. It is concluded that Ca2+ in physiological concentrations, as a second messenger, can control phosphorylation of the low molecular mass phospoproteins in RBM, in addition to well known regulation of some Krebs cycle dehydrogenases by Ca2+. The protein phosphorylation was strongly dependent on the Ca2+-induced PTP opening.     

Phosphorylation in sealed rod outer segments: effects of cyclic nucleotides

Rod outer segments (ROS) from rat were purified on Percoll gradients. These ROS had intact plasma membranes since they were impermeable to small molecules. Protein phosphorylation in the purified ROS was studied after the plasma membrane was disrupted by freeze/thawing. [gamma-32P]ATP was used as phosphate donor. ATP concentration, time, temperature, and light or dark adaptation were varied in the assays. The 32P-labeled proteins were separated by polyacrylamide gel electrophoresis and autoradiographed. Rhodopsin was the dominant phosphorylated protein, and the addition of adenosine cyclic 3',5'-phosphate (cAMP) or guanosine cyclic 3',5'-phosphate (cGMP) (10(-4) M) did not qualitatively alter the ROS phosphorylation pattern. The only cyclic nucleotide effect we could establish in these experiments was the inhibition of rhodopsin phosphorylation by cGMP. This inhibition did not appear to be competitive with ATP since cAMP was much less inhibitory than cGMP and the phosphorylation in the presence of cGMP reached a plateau at a much lower level than in control conditions. Hypotheses implying an involvement of protein phosphorylation/dephosphorylation in dark adaptation have been formulated [Miller, J. A., & Paulsen, R. (1975) J. Biol. Chem. 250, 4427-4432; Kuhn, H., McDowell, J. H., Leser, K. H., & Bader, S. (1977) Biophys. Struct. Mech. 3, 175-180]; we suggest that cGMP may control this process through the modulation of the extent of inhibition of phosphorylation of the visual pigment.     

Rat growth hormone-releasing factor stimulates cyclic GMP formation and phosphatidylinositol metabolism in the median eminence.

The effects of rat growth hormone releasing factor (rGRF) on somatostatin (SRIF) secretion, cyclic nucleotide production and phosphatidylinositol metabolism were investigated in the median eminence (ME), using an in vitro system. Medium was discarded and replaced by medium containing various concentrations of rGRF or rGRF plus epinephrine (E, 6 x 10(-7) M). rGRF had no effect on basal or E-stimulated release of cAMP. In the same experiments rGRF markedly stimulated SRIF release. These results suggested that cAMP is not involved in the stimulatory effect of GRF on SRIF release. However, GRF significantly stimulated release of both SRIF and cGMP in a dose-related manner. Maximal stimulation was observed at 10(-10) M GRF (p less than 0.005) which also produces maximal SRIF release. 2'0-monobutyrylguanosine 3'5' cyclic phosphate (mbcGMP, 10(-11) to 10(-10) M) stimulated SRIF release from ME fragments (p less than 0.001 at 10(-10) M) whereas the control, sodium butyrate (10(-6) M), had no effect. GRF caused significant elevation of 30.6% in the concentration of labelled inositol phosphates [( 3H]-IPs) in the ME. These data indicate that GRF stimulation of SRIF release is accompanied by increased cGMP production and phosphatidyl-inositol (PI) metabolism but does not alter cAMP production. Because mbcGMP can directly stimulate SRIF release, we suggest that GRF causes a receptor-mediated increase in the metabolism of phosphatidylinositol and cGMP formation. These actions therefore may be among the early metabolic events in the mechanism of GRF-stimulated SRIF release from the ME.     

Protein phosphorylation in intact coronary endothelial cells by bradykinin

In cultured coronary endothelial cells obtained from guinea pig hearts, bradykinin (10(-6) M) stimulated the 32Pi-incorporation into 5 substrate proteins with molecular weights corresponding to 27, 32, 60, 86 and 100 kDa. The time course of phosphorylation of the 60, 86 and 100 kDa proteins was rapid (within 30 s), but transient (max. within 1-2 min.), while the 32Pi incorporation into the 27 and 32 kDa protein was delayed but increased within 10 minutes. Ca+(+)-ionophore A 23187 (10(-5) M) and 12-O-tetradecanoylphorbol-13-acetate (TPA) (10(-5) M) both mimicked the effects of the bradykinin induced phosphorylation pattern. While A 23187 enhanced the phosphorylation of the 27, 60 and 100 kDa substrates, TPA increased the 32Pi-incorporation into the 32 and 86 kDa proteins. Furthermore the time course of protein phosphorylation elicited by A 23187 and TPA showed marked similarities to those obtained with bradykinin. Our findings are consistent with the view, that stimulation of coronary endothelial bradykinin-receptors activates both Ca+(+)-dependent protein kinases and protein kinase C.     

Cyclic nucleotide-dependent endogenous phosphorylation of rabbit myometrium membranes

The membrane-bound protein kinase activity in plasma membranes (PM) and sarcoplasmic reticulum (SR) of rabbit myometrium was revealed, which catalyzes the synthesis of protein phosphoester products. cAMP had no effect on the phosphorylation of membrane substrates by soluble protein kinases I and II as well as by the membrane-bound enzyme of SR. At the same time, cAMP (10(-8) stimulated by 200% the phosphorylation of sarcolemmal components at functional rest (FR). In preparations obtained from pregnant animals, cAMP (10(-8) and 10(-5) M) stimulated the phosphorylation of PM 7- and 3-fold, respectively. cGMP had no effect on the phosphorylation of PM and SR proteins at FR. At 10(-5) and 10(-8) M, cGMP stimulated endogenous phosphorylation of PM and SR 7- and 4-fold, respectively. In pregnancy, the degree of endogenous phosphorylation of PM and SR increased by 70% and 260% as compared to that at FR; the activity of soluble protein kinases decreased two times under these conditions. At FR, the sarcolemmal proteins with Mr 35 000, 57 000, 89 000 and 174 000 underwent phosphorylation. The phosphorylation of the proteins with Mr 35 000 and 57 000 was cAMP-dependent. In pregnant animals sarcolemma, the phosphorylation affected the proteins with Mr 47 000, 57 000 and 174 000 and was cAMP-dependent for the former two proteins and cGMP-dependent for the latter protein. At FR, two SR proteins with Mr 47 000 and 168 000, while in pregnant animals the proteins with Mr 47 000, 132 000 and 168 000 were phosphorylatable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Atrial natriuretic factor inhibits autophosphorylation of protein kinase C and A 240-kDa protein in plasma membranes of bovine adrenal glomerulose cells: Involvement of cGMP-dependent and independent signal transduction mechanisms

To clarify the intracellular signalling mechanisms of atrial natriuretic factor (ANF), we studied its effect on protein phosphorylation in plasma membranes of bovine adrenal cortical cells. ANF (1×10^–7 M) inhibited phosphorylation of the 78-kDa protein kinase C (PKC) and a 240-kDa protein in specific manner. In parallel experiments, cGMP (0.5 mM) inhibited phosphorylation of only the 78-kDa PKC but it did not affect phosphorylation of the 240-kDa protein. Phosphorylation of the 78-kDa PKC was enhanced in a Ca²⁺-/phospholipid-dependent manner. However, after prolonged preincubation of plasma membranes with Ca²⁺ (0.5 mM), the incorporation of³²P-radioactivity rapidly decreased in the 78-kDa PKC and subsequently increased in the 45- and 48-kDa protein bands due to Ca²⁺-dependent proteolytic degradation of 78-kDa PKC. Polyclonal antibodies against brain PCK were used to immunoblot and immunoprecipitate the 78-kDa PKC. Preincubation of plasma membranes with Ca²⁺ for varying times, followed by immunoblotting revealed a gradual loss of the immunoreactive 78-kDa PKC band in a time-dependent manner. Immunoprecipitation of phosphorylated 78-kDa PKC in plasma membranes showed that its phosphorylation was significantly inhibited in the presence of ANF as compared to control membranes, phosphorylated in the absence of ANF. The results in this present study document a new signal transduction mechanism of ANF at molecular level which possibly involves dephosphorylation of the 78-kDa PKC and a 240-kDa protein in a cGMP-dependent and-independent manner in bovine adrenal glomerulosa cell membranes. (Mol Cell Biochem141: 103–111, 1994)     

Regulation of cGMP production by intracellular alkalinization in cultured rat inner medullary collecting duct cells

We evaluated the relationship between cell pH and cGMP production in cultured rat renal inner medullary collecting duct cells. The cGMP level, 21 +/- 6, was not different in control vs. alkalinized cells, 49 +/- 17 fmol/mg protein (p greater than 0.5). 10(-11) M atrial natriuretic peptide (ANF) enhanced cGMP production in alkalinized cells, 426 +/- 34 vs. 141 +/- 9*. Conversely, alkalinization inhibited 10(-4)M nitroprusside (SNP) induced cGMP formation, 29 +/- 9 vs. 332 +/- 67*. Phosphodiesterase inhibition abolished the difference in cGMP production by ANF but did not reverse the inhibitory effect of alkalinization on SNP induced cGMP production. In rat renal inner medullary collecting duct cells, cellular alkalinization plays a significant role in the regulation of guanylate cyclase mediated cGMP production. * = p less than 0.05).     

Nonequilibrium kinetics of a cyclic GMP-binding protein in dictyostelium discoideum

Chemoattractants added to cells of the cellular slime mold dictyostelium discoideum induce a transient elevation of cyclic GMP levels, with a maximum at 10 s and a recovery of basal levels at approximately 25 s after stimulation. We analyzed the kinetics of an intracellular cGMP binding protein in vitro and in vivo. The cyclic GMP binding protein in vitro at 0 degrees C can be described by its kinetic constants K(1)=2.5 x 10(6) M(- 1)s(-1), k(-1)=3.5 x 10(-3)s(-1), K(d)=1.4 x 10(-9) M, and 3,000 binding sites/cell. In computer simulation experiments the occupancy of the cGMP binding protein was calculated under nonequilibrium conditions by making use of the kinetic constants of the binding protein and of the shape of the cGMP accumulations. These experiments show that under nonequilibrium conditions by making use of the kinetic constants of the binding protein and the shape of the cGMP accumulations. These experiments show that under nonequilibrium conditions the affinity of the binding protein for cGMP is determined by the rate constant of association (k(1)) and not by the dissociation constant (k(d)). Experiments in vivo were performed by stimulation of aggregative cells with the chemoattractant cAMP, which results in a transient cGMP accumulation. At different times after stimulation with various cAMP concentrations, the cells were homogenized and immediately thereafter the number of binding proteins which were not occupied with native cGMP were determined. The results of these experiments in vivo are in good agreement with the results of the computer experiments. This may indicate that: (a) The cGMP binding protein in vivo at 22 degrees C can be described by its kinetic constants: K(1)=4x10(6)M(-1)s(-1) and K(-1)=6x10(-3)s(-1). (b) Binding the cGMP to its binding protein is transient with a maximum at about 20-30 s after chemotactic stimulation, followed by a decay to basal levels, with a half-life of approximately 2 min. (c) The cGMP to its binding proteins get half maximally occupied at a cGMP accumulation of δ[cGMP](10)=2x10(-8) M, which corresponds to an extracellular stimulation of aggregative cells by 10(-10) M cAMP. (d) Since the mean basal cGMP concentration is approximately 2x10(-7) M, the small increase of cGMP cannot be detected accurately. Therefore the absence of a measurable cGMP accumulation does not argue against a cGMP function. (e) There may exist two compartments of cGMP: one contains almost all the cGMP of unstimulated cells, and the other contains cGMP binding proteins and the cGMP which accumulates after chemotactic stimulation. (f) From the kinetics of binding, the cellular responses to the chemoattractant can be divided into two classes: responses which can be mediated by this binding protein (such as light scattering, proton extrusion, PDE induction, and chemotaxis) and responses which cannot be (solely) mediated by this binding protein such as rlay, refractoriness, phospholipids methylation, and protein methylation.     

Effects of atrial natriuretic factor, nitroprusside and acetylcholine on cGMP immunostaining in the isolated perfused rat kidney.

In the present study the localization of the cGMP production in response to the vasodilators acetylcholine (ACh) and sodium nitroprusside (SNP) and to atrial natriuretic factor (ANF) was studied in the isolated perfused rat kidney using cGMP immunocytochemistry. After ACh (0.3 microM) infusion increased cGMP immunoreactivity was found in kidney interlobar and segmental arteries and in glomeruli. SNP (1 microM) and ANF (0.01 microM) elevated cGMP staining in the same elements of the kidney as ACh. In the glomeruli ACh and SNP stimulated cGMP production in mesangial cells whereas ANF stimulated cGMP production in mesangial cells whereas ANF stimulated cGMP production in epithelial cells (podocytes). However, SNP at higher doses (10 microM) stimulated cGMP production not only in glomeruli, but also in interstitial cells throughout the cortex. In addition SNP and ANF increased cGMP production in the medulla.     

Atrial natriuretic factor regulates steroidogenic responsiveness and cyclic nucleotide levels in mouse Leydig cells in vitro

The effects of synthetic atrial natriuretic factor (ANF) on the regulation of mouse Leydig cell steroidogenesis have been studied in vitro. ANF in nanomolar concentration increased testosterone production by more than 30-fold over basal levels. Concomitantly, cyclic guanosine monophosphate levels were increased 35-fold; cyclic adenosine monophosphate levels fell minimally (15-20%). ANF at low concentration (1 X 10(-11) M) inhibited testosterone production by luteinizing hormone-stimulated cells, while at higher concentration (greater than 2 X 10(-9) M) ANF stimulated steroidogenesis beyond the level attained by luteinizing hormone alone. These results indicate that ANF can exert stimulatory effects on testosterone steroidogenesis in vitro, and that the mechanism may involve an intracellular messenger other than cyclic adenosine monophosphate.