A1 and A2 adenosine receptors in rabbit cortical collecting tubule cells. Modulation of hormone-stimulated cAMP.

Adenosine analogs were used to investigate the cellular mechanisms by which adenosine may alter renal tubular function. Cultured rabbit cortical collecting tubule (RCCT) cells, isolated by immunodissection, were treated with 5'-N-ethylcarboxamideadenosine (NECA), N6-cyclohexyladenosine (CHA), and R-N6-phenylisopropyladenosine (PIA). All three analogs produced both dose-dependent inhibition and stimulation of RCCT cell cyclic AMP (cAMP) production. Stimulation of cAMP accumulation occurred at analog concentrations of 0.1 microM to 100 microM with the rank order of potency NECA greater than PIA greater than CHA. Inhibition occurred at concentrations of 1 nM to 1 microM with the rank order of potency CHA greater than PIA greater than NECA. These effects on cAMP production were inhibited by 1,3-diethyl-8-phenylxanthine and isobutylmethylxanthine. CHA (50 nM) blunted AVP- and isoproterenol-stimulated cAMP accumulation. This modulation of hormone-induced cAMP production was abolished by pretreatment of RCCT cells with pertussis toxin. Prostaglandin E2 production was unaffected by 0.1 mM CHA. These findings indicate the presence of both inhibitory (A1) and stimulatory (A2) receptors for adenosine in RCCT cells. Moreover, occupancy of the A1 receptor causes inhibition of both basal and hormone-stimulated cAMP formation through an action on the inhibitory guanine nucleotide-binding regulatory component, Ni, of the adenylate cyclase system.

Concentrations of prostaglandin endoperoxide synthase and prostaglandin I2 synthase in the endothelium and smooth muscle of bovine aorta.

Platelets adhere to the subendothelial layer of newly deendothelialized arteries. Attachment can be reduced with exogenous prostacyclin (PGI2). Thus, the subendothelium may be unable to produce sufficient PGI2 to prevent platelet adherence and subsequent platelet-platelet interaction. Consistent with this explanation are data from an earlier report (1977. Moncada S., A. G. Herman, E. A. Higgs, and J. R. Vane. Thromb. Res. 11:323-344) indicating that the smooth muscle layer of aorta has only 10-15% of the capacity of endothelial cells to synthesize PGI2. We have measured the concentrations of PGI2 synthase and prostaglandin endoperoxide (PGH) synthase in bovine aorta and obtained results quite different from those described in this earlier report. Tandem immunoradiometric assays for PGI2 synthase and PGH synthase antigens were used to quantitate these proteins in detergent-solubilized homogenates of endothelial cells and smooth muscle tissue prepared from 10 different bovine aorta. The concentrations of PGI2 synthase in endothelial cells and smooth muscle were found to be the same. However, the concentration of PGH synthase in endothelial cells averaged greater than 20 times that of smooth muscle. Results similar to those determined by immunoradiometric assay were also obtained when PGH synthase and PGI2 synthase catalytic activities were measured in preparations of endothelial and smooth muscle cells. Furthermore, when bovine aorta and renal arteries were subjected to immunocytofluorescence staining using monoclonal antibodies to PGI2 synthase, fluorescence staining of equivalent intensity was detected in both the endothelial cells and the smooth muscle. Moreover, the intensity of fluorescence was similar throughout cross-sections of vascular smooth muscle, indicating that there is no gradient in PGI2 synthase concentrations between the endothelium and adventitia. Our results indicate that the propensity of platelets to adhere to the subendothelium of deendothelialized arteries and form aggregates cannot be attributed simply to an inability of the denuded vasculature to produce PGI2 from PGH2, but may be a consequence of the low PGH synthase activity of smooth muscle. Consistent with this concept are the results of Eldor et al. (1981. J. Clin. Invest. 67:735-741) who reported that increases in PGH synthase activity are associated with formation of a nonthrombogenic neointima.

Calmodulin-stimulated cyclic nucleotide phosphodiesterase (PDE1C) is induced in human arterial smooth muscle cells of the synthetic, proliferative phenotype.

The diversity among cyclic nucleotide phosphodiesterases provides multiple mechanisms for regulation of cAMP and cGMP in the cardiovascular system. Here we report that a calmodulin-stimulated phosphodiesterase (PDE1C) is highly expressed in proliferating human arterial smooth muscle cells (SMCs) in primary culture, but not in the quiescent SMCs of intact human aorta. High levels of PDE1C were found in primary cultures of SMCs derived from explants of human newborn and adult aortas, and in SMCs cultured from severe atherosclerotic lesions. PDE1C was the major cAMP hydrolytic activity in these SMCs. PDE expression patterns in primary SMC cultures from monkey and rat aortas were different from those from human cells. In monkey, high expression of PDE1B was found, whereas PDE1C was not detected. In rat SMCs, PDE1A was the only detectable calmodulin-stimulated PDE. These findings suggest that many of the commonly used animal species may not provide good models for studying the roles of PDEs in proliferation of human SMCs. More importantly, the observation that PDE1C is induced only in proliferating SMCs suggests that it may be both an indicator of proliferation and a possible target for treatment of atherosclerosis or restenosis after angioplasty, conditions in which proliferation of arterial SMCs is negatively modulated by cyclic nucleotides.

Isolation and characterization of human cDNAs encoding a cGMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase.

Human cyclic GMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase (PDE2A3) cDNAs were cloned from hippocampus and fetal brain cDNA libraries. A 4.2-kb composite DNA sequence constructed from overlapping cDNA clones encodes a 941 amino acid protein with a predicted molecular mass of 105,715 Da. Extracts prepared from yeast expressing the human PDE2A3 hydrolyzed both cyclic AMP (cAMP) and cyclic GMP (cGMP). This activity was inhibited by EHNA, a selective PDE2 inhibitor, and was stimulated three-fold by cGMP. Human PDE2A is expressed in brain and to a lesser extent in heart, placenta, lung, skeletal muscle, kidney and pancreas. The human PDE2A3 differs from the bovine PDE2A1 and rat PDE2A2 proteins at the amino terminus but its amino-terminal sequence is identical to the bovine PDE2A3 sequence. The different amino termini probably arise from alternative exon splicing of the PDE2A mRNA.

Isolation and characterization of cDNAs encoding PDE5A, a human cGMP-binding, cGMP-specific 3',5'-cyclic nucleotide phosphodiesterase.

Human cGMP-binding, cGMP-specific 3',5'-cyclic nucleotide phosphodiesterase (PDE5A) cDNAs were isolated. A 3.1-kb composite DNA sequence assembled from overlapping cDNAs encodes an 875-amino-acid protein with a predicted molecular mass of 100012 Da (PDE5A1). Extracts prepared from yeast expressing human PDE5A1 hydrolyzed cGMP. This activity was inhibited by the selective PDE5 inhibitors zaprinast and DMPPO. PDE5A mRNA is expressed in aortic smooth muscle cells, heart, placenta, skeletal muscle and pancreas and, to a much lesser extent, in brain, liver and lung. A 5'-splice variant, PDE5A2, encodes an 833-amino-acid protein with eight unique amino acids at the amino terminus. PDE5A maps to chromosome 4q 25-27.

The biosynthesis and actions of prostaglandins in the renal collecting tubule and thick ascending limb.

PGE2 formed by renal collecting tubules is an important factor in regulating NaCl and water reabsorption in the collecting tubule and medullary thick ascending limb. PGE2 appears to act, depending on its ambient concentration, via several different receptors present in these renal epithelia to modulate cAMP turnover in both positive and negative directions. These putative PGE receptors form a family of receptors, all coupled to G proteins, and this family of PGE receptors is homologous to the adrenergic receptor family.

Cyclic GMP and regulation of cyclic nucleotide hydrolysis.

Several of the different PDE isozyme families have the ability in vitro to hydrolyze cGMP. In particular they include the CaM-dependent PDEs, the cGMP-stimulated PDEs, and the cGMP binding, cGMP-specific PDEs. Existing evidence suggests or demonstrates that in different cell types, each of these can be important determinants for the control of cGMP steady-state levels. Each of these enzymes is differentially expressed and regulated; moreover, the amount of the enzyme expressed and the mode of regulation determine to a large extent the rate of rise, maximal level, rate of fall, and duration of the cGMP signal in the cell. In addition to enzymes that function to degrade cGMP at least two also are regulated by cGMP both in vitro and in the intact cell. The cGMP-stimulated PDE has the ability to decrease cAMP levels in response to cGMP and the cGMP-inhibited PDE can increase cAMP levels in response to cGMP. We are just beginning to define how many different isozymes of PDE exist in mammalian tissues, where they are located, and how they are regulated. Selective inhibitors to each are being developed and studies designed to define structural features that determine the mechanisms of action and regulation of the PDEs have been initiated. It is expected that in the next few years more PDEs will be discovered and the functions of the new an existing ones with be more clearly defined.

Regulation of cyclic AMP metabolism in rabbit cortical collecting tubule cells by prostaglandins.

Prostaglandin E1 (PGE1) at 1 nM inhibits arginine-vasopressin (AVP)-induced water reabsorption in the rabbit cortical collecting tubule (RCCT), while 100 nM PGE1, by itself, stimulates water reabsorption (Grantham, J. J., and Orloff, J. (1968) J. Clin. Invest. 47, 1154-1161). To investigate the basis for these two responses, we measured the effects of prostaglandins on cAMP metabolism in purified RCCT cells. In freshly isolated cells, PGE2, PGE1, and 16,16-dimethyl-PGE2 acting at high concentrations (0.1-10 microM) stimulated cAMP accumulation; however, one PGE2 analog, sulprostone (16-phenoxy-17,18,19,20-tetranor-PGE2 methylsulfonilamide), failed to stimulate cAMP accumulation or to antagonize PGE2-induced cAMP formation; PGD2, PGF2 alpha, and a PGI2 analog, carbacyclin (6-carbaprostaglandin I2), also failed to stimulate cAMP synthesis. These results suggest that there is a PGE-specific stimulatory receptor in RCCT cells which mediates activation of adenylate cyclase. Occupancy of this receptor would be anticipated to cause water reabsorption by the collecting tubule. At lower concentrations (0.1-100 nM) PGE2, PGE1, 16,16-dimethyl-PGE2, and, in addition, sulprostone inhibited AVP-induced cAMP accumulation by fresh RCCT cells in the presence of cAMP phosphodiesterase inhibitors. Pertussis toxin pretreatment of RCCT cells blocked the ability of both PGE2 and sulprostone to inhibit AVP-induced cAMP accumulation. In membranes prepared from RCCT cells, sulprostone prevented stimulation of adenylate cyclase by AVP. These results suggest that E-series prostaglandins (including sulprostone) can act through an inhibitory PGE receptor(s) coupled to the inhibitory guanine nucleotide regulatory protein, Gi, to block AVP-induced cAMP synthesis by RCCT cells. Occupancy of this receptor would be expected to cause inhibition of AVP-induced water reabsorption in the intact tubule. Curiously, after RCCT cells were cultured for 5-7 days, PGE2 no longer inhibited AVP-induced cAMP accumulation, but PGE2 by itself could still stimulate cAMP accumulation. In contrast to PGE2, epinephrine acting via an alpha 2-adrenergic, Gi-linked mechanism did block AVP-induced cAMP formation by cultured RCCT cells. This implies that some component of the inhibitory PGE response other than Gi is lost when RCCT cells are cultured.

Molecular cloning of a cDNA encoding the "61-kDa" calmodulin-stimulated cyclic nucleotide phosphodiesterase. Tissue-specific expression of structurally related isoforms.

We have isolated a 2287-bp cDNA encoding the 61-kDa calmodulin-stimulated cyclic nucleotide phosphodiesterase (CaM PDE) from a bovine brain library. A large open reading frame within the cDNA encodes a 530-residue polypeptide which is identical to the sequence of the purified protein previously determined by direct amino acid sequencing. Moreover, COS cells transfected with the cDNA express a cAMP and cGMP hydrolytic activity that is stimulated by calcium and calmodulin, confirming that the cDNA represents a mRNA species encoding a CaM PDE isozyme. RNase protection analyses indicate that either 61-kDa CaM PDE mRNA or structurally related transcripts encoding different CaM PDE isoforms are expressed in a tissue-specific manner. Total RNA isolated from brain (cerebral cortex, basal ganglia, hippocampus, cerebellum, and medulla/spinal cord), heart, aorta, liver, kidney outer medulla, kidney papilla, trachea, and lung completely protected a 410-base antisense riboprobe corresponding to sequence encoding a portion of the catalytic domain. Little or no protection was detected using adrenal cortex, adrenal medulla, liver, kidney cortex, spleen, or T-lymphocyte total RNA. Only brain RNA completely protected a 240-base antisense riboprobe corresponding to the 61-kDa CaM PDE amino terminus encompassing a putative calmodulin-binding domain. However, heart, aorta, liver, kidney, trachea, and lung RNA protected 150 bases of this riboprobe suggesting that these tissues express an isoform structurally related to the 61-kDa CaM PDE. Northern analysis of mRNA isolated from brain, heart, aorta, liver, kidney, lung, and trachea revealed that the cDNA hybridizes with a 3.8- and a 4.4-kb (kilobase) mRNA species. Interestingly, Northern blots of bovine cerebral cortex and heart mRNA probed under stringent conditions with antisense transcripts corresponding to either the 5'- or 3'-untranslated sequence of the 61-kDa CaM PDE cDNA hybridized with only the 4.4-kb mRNA from both tissues. Since different, yet structurally similar CaM PDE isoforms are expressed in brain and in heart, this result, in addition to the RNase protection data, is consistent with the idea that the mRNAs encoding these two CaM PDE isoforms are products of an alternately spliced gene.

The role of protein phosphorylation in the regulation of cyclic nucleotide phosphodiesterases.

The cyclic nucleotide phosphodiesterases constitute a complex superfamily of enzymes responsible for catalyzing the hydrolysis of cyclic nucleotides. Regulation of cyclic nucleotide phosphodiesterases is one of the two major mechanisms by which intracellular cyclic nucleotide levels are controlled. In many cases the fluctuations in cyclic nucleotide levels in response to hormones is due to the hormone responsiveness of the phosphodiesterase. Isozymes of the cGMP-inhibited, cAMP-specific, calmodulin-stimulated and cGMP-binding phosphodiesterases have been demonstrated to be substrates for protein kinases. Here we review the evidence that hormonally responsive phosphorylation acts to regulate cyclic nucleotide phosphodiesterases. In particular, the cGMP-inhibited phosphodiesterases, which can be phosphorylated by at least two different protein kinases, are activated as a result of phosphorylation. In contrast, phosphorylation of the calmodulin-stimulated phosphodiesterases, which coincides with a decreased sensitivity to activation by calmodulin, results in decreased phosphodiesterase activity.

A prostaglandin E receptor coupled to a pertussis toxin-sensitive guanine nucleotide regulatory protein in rabbit cortical collecting tubule cells.

At different concentrations, prostaglandin E2 (PGE2) can either stimulate or inhibit cAMP formation in freshly isolated rabbit cortical collecting tubule (RCCT) cells, but in cultured RCCT cells PGE2 can only stimulate cAMP synthesis (Sonnenburg, W. K., and Smith W. L. (1989) J. Biol. Chem. 263, 6155-6160). Here, we report characteristics of [3H]PGE2 binding to membrane receptor preparations from both freshly isolated and cultured RCCT cells. [3H]PGE2 binding to membranes from freshly isolated RCCT cells was saturable and partially reversible. Equilibrium binding analyses indicated that in the absence of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) there is a single class of PGE2 binding sites (KD = 4.2 +/- 0.4 nM; Bmax = 583 +/- 28 fmol/mg); in the presence of 100 microM GTP gamma S, there is also only one class of binding sites but with a somewhat lower KD = 1.2 +/- 0.5 nM (Bmax = 370 +/- 40 fmol/mg). This stimulatory effect of GTP gamma S was blocked by pretreatment of the freshly isolated RCCT cells with pertussis toxin. The relative affinities of prostanoids for the [3H]PGE2-binding site were determined to be 17,18,19,20-tetranor-16-phenoxy-PGE2-methylsulfonylamide (sulprostone) approximately PGE2 approximately PGE1 approximately 16,16-dimethyl-PGE2 greater than carbacyclin approximately PGF2 alpha greater than PGD2. This is the order of potency with which prostaglandins inhibit arginine vasopressin-induced cAMP formation in fresh RCCT cells. Interestingly, [3H]PGE2 binding to membranes from cultured cells, which, unlike fresh cells, fail to show an inhibitory response to PGE2, was only 10-20% of that observed with membranes from fresh cells; moreover, binding of [3H]PGE2 to membranes from cultured cells was neither stimulated by GTP gamma S nor inhibited by sulprostone. The prostanoid binding specificities and the unusual pertussis toxin-sensitive, stimulatory effect of GTP gamma S on binding of [3H]PGE2 to membranes from freshly isolated RCCT cells are characteristics shared by a Gi-linked PGE receptor from renal medulla (Watanabe, T., Umegaki, K., and Smith, W. L. (1986) J. Biol. Chem. 261, 14340-14349). Our results suggest that the [3H]PGE2 binding site of freshly isolated RCCT cells is the PGE receptor which is coupled to a Gi to attenuate arginine vasopressin-induced cAMP synthesis in the renal collecting tubule.

Molecular cloning of a cyclic GMP-stimulated cyclic nucleotide phosphodiesterase cDNA. Identification and distribution of isozyme variants.

We have cloned a 4.2-kilobase pair (kb) cDNA that encodes the cyclic GMP-stimulated phosphodiesterase (cGS PDE) from a bovine adrenal cortex library. The 921-residue polypeptide deduced from the cDNA nucleotide sequence is nearly identical with the complete amino acid sequence of the cGS PDE purified from a soluble bovine heart extract. Moreover, PPD-S49 cells transfected with the cGS PDE cDNA express a soluble cAMP hydrolytic activity that is enhanced by cGMP. Total RNA isolated from several bovine tissues were screened for cGS PDE transcript by Northern blot analysis. The cGS PDE cDNA appears to hybridize to a single 4.5-4.6-kb mRNA species. Although the cGS PDE mRNA is most abundant in the adrenal cortex, it is also concentrated in the adrenal medulla and heart and in anatomically distinct regions of the brain and kidney. A mRNA species encoding a putative variant cGS PDE isoform was detected by RNase protection. Total RNA isolated from adrenal cortex, adrenal medulla, liver, kidney, trachea, lung, spleen, and T-lymphocytes completely protected a 452-base riboprobe encoding 100 residues of the adrenal cortex cGS PDE amino terminus. In contrast, RNAs isolated from brain (cerebral cortex, hippocampus, and basal ganglia) protected only 268 bases of this riboprobe. The RNase protection pattern of this same probe using heart RNA showed major bands at both 268 and 452 bases, suggesting that two different cGS PDE mRNA species are expressed. These results indicate that the cGS PDE is widely expressed in a variety of tissues. Moreover, these studies suggest that at least one different cGS PDE isoform having a structurally distinct amino-terminal domain is expressed in brain and heart.

Regulation of cAMP metabolism by PGE2 in cortical and medullary thick ascending limb of Henle's loop.

We have examined the regulation by prostaglandin E2 (PGE2) of hormone-induced adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in cells isolated by immunodissection from both the medullary and cortical thick ascending limb of Henle's loop of rabbit kidney. At concentrations greater than 10(-8) M, PGE2, but not sulprostone (16-phenoxy-17,18,19,20-tetranor-PGE2 methylsulfonilamide), caused cAMP accumulation in both cortical and medullary thick limb cells. However, at concentrations of less than or equal to 10(-8) M, both PGE2 and sulprostone inhibited arginine vasopressin (AVP)-, calcitonin-, and glucagon-induced cAMP accumulation in medullary thick ascending limb (mTAL) cells. In cortical thick limb (cTAL) cells, sulprostone also inhibited AVP-, calcitonin-, and parathyroid hormone (PTH)-induced cAMP accumulation. The inhibitory effects of PGE2 and of sulprostone were blocked by pretreatment of mTAL and cTAL cells with pertussis toxin. Membranes prepared from mTAL cells exhibited a [3H]PGE2 binding activity that was stimulated on addition of the stable guanosine 5'-triphosphate (GTP) analogue, 5'-guanosine gamma-thiotriphosphate (GTP gamma S); moreover, sulprostone inhibited [3H]PGE2 binding. Our results suggest that PGE2 can function via a prostaglandin E receptor linked to a guanine nucleotide regulatory protein, Gi, to attenuate hormone-induced cAMP formation in both mTAL and cTAL cells of rabbit kidney.

Differential expression of the 61 kDa and 63 kDa calmodulin-dependent phosphodiesterases in the mouse brain.

Based on their relative abundance and regulation by Ca2+ and by phosphorylation in vitro, it is thought that the Ca2+/calmodulin-dependent phosphodiesterases (CaM-PDEs) are important modulators of cyclic nucleotide function in the brain. Two of the most abundant CaM-PDEs in the brain are the 61 kDa and 63 kDa isozymes. In this study, the regional and cellular expression of mRNA encoding these two different isoforms in mouse brain has been determined by in situ hybridization. The 63 kDa CaM-PDE mRNA has a wide-spread but uneven distribution. Very strong hybridization signals are present in the caudate-putamen, nucleus accumbens, olfactory tubercle, and dentate gyrus of the hippocampus. Somewhat lesser amounts of 63 kDa CaM-PDE mRNA are present in the olfactory bulb and piriform cortex. Weaker but still easily discernible hybridization signals are seen in several layers of the cerebral cortex, CA1 and CA3 regions of the hippocampus, amygdaloid nuclear complex, thalamus, hypothalamus, midbrain, brainstem, cerebellum, and spinal cord. A weak hybridization signal was detected in the globus pallidus of the basal ganglia. In general, the distribution of the 63 kDa CaM-PDE is very similar to that of dopamine receptors, suggesting that it may modulate dopamine function. In contrast, the 61 kDa CaM-PDE mRNA has a more limited and much different distribution, with the highest level of expression in the cerebral cortex and in the pyramidal cells of the hippocampus. A moderate hybridization signal was detected in the medial habenula and amygdaloid nuclear complex. In addition, small subsets of neurons in several other regions showed specific hybridization. Both PDE mRNAs appear to be localized exclusively in neuronal cell bodies. Their distinct distribution suggests important but different physiological roles for these two isozymes in the regional regulation of cyclic nucleotides in the CNS. Since these two isozymes are differentially phosphorylated by cAMP-dependent and Ca2+/CaM-dependent protein kinases, the differential expression also provides a potential mechanism by which these PDEs can differentially regulate cAMP and cGMP in different brain areas. The high expression levels in specific subsets of neurons also suggest that agents increasing Ca2+ in these neurons will increase the rate of cyclic nucleotide degradation.

Stage and cell-specific expression of calmodulin-dependent phosphodiesterases in mouse testis.

Calcium and cyclic nucleotides are second messengers that regulate the development and functional activity of spermatozoa. Calcium/calmodulin-dependent phosphodiesterases (CaM-PDEs) are abundant in testicular cells and in mature spermatozoa and provide one means by which calcium regulates cellular cyclic nucleotide content. We examined the spatial and temporal expression profiles of three knownCaM-PDE genes, PDE1A, PDE1B, and PDE1C, in the testis. In situ hybridization and immunofluorescent staining showed that both PDE1A and PDE1C are highly expressed but at different stages in developing germ cells. However, a very low hybridization signal of PDE1B exists uniformly throughout the seminiferous epithelium and the interstitium. More specifically, PDE1A mRNA is found in round to elongated spermatids, with protein expression in the tails of elongated and maturing spermatids. In contrast, PDE1C mRNA accumulates during early meiotic prophase and throughout meiotic and postmeiotic stages. Immunocytochemistry showed a diffuse, presumably cytosolic distribution of the expressed protein. The distinct spatial and temporal expression patterns of CaM-PDEs suggest important but different physiological roles for these CaM-PDEs in developing and mature spermatozoa.

Immunodissection of cortical and medullary thick ascending limb cells from rabbit kidney.

A procedure was developed for isolating thick ascending limb cells from either the outer medulla or the inner cortex from rabbit kidneys. Dispersed cells derived from the medulla or cortex were incubated with goat anti-human uromucoid (Tamm-Horsfall glycoprotein) serum, washed, and applied to culture dishes coated with affinity-purified anti-goat immunoglobulin G. Nonadherent cells were removed by washing. Routinely, 10(6) or 7 X 10(4) adherent cells were obtained per gram of rabbit outer medulla or inner cortex, respectively. Greater than 97% of the adherent cells stained for Tamm-Horsfall antigen, and examination of freshly isolated cells by transmission electron microscopy established that they had morphological properties expected for thick limb cells. Freshly isolated medullary thick limb (MTALH) cells consistently accumulated cAMP in response to arginine vasopressin (AVP), thyrocalcitonin, prostaglandin E2 (PGE2), and glucagon. PGE2, thyrocalcitonin, parathyroid hormone, and AVP, but not isoproterenol or glucagon, reproducibly stimulated cAMP accumulation in freshly isolated cortical thick limb (CTALH) cells. MTALH cells produced immunoreactive PGE2 when incubated with 10 microM arachidonic acid. In summary, large numbers of highly purified and hormonally responsive rabbit MTALH and CTALH cells can be obtained by immunodissection using commercially available antibody preparations. Because the Tamm-Horsfall antigen is present as an extracellular determinant on thick ascending limb epithelia from many species, this general approach likely can be used to isolate CTALH and MTALH cells from most mammalian kidneys.

Immunodissection and culture of rabbit cortical collecting tubule cells.

A mouse monoclonal antibody designated IgG3(rct-30) has been prepared that reacts specifically with an antigen on the surface of all cells comprising the cortical and medullary rabbit renal collecting tubule including the arcades. Plastic culture dishes coated with IgG3(rct-30) were used to isolate collecting tubule cells from collagenase dispersions of rabbit renal cortical cells by immunoadsorption. Typically, 10(6) rabbit cortical collecting tubule (RCCT) cells were obtained from 5 g of renal cortex (2 kidneys). Initial purity was greater than 96% based on immunocytofluorescent staining with three different anti-collecting tubule antibodies. Between 20 and 30% of the RCCT cells were reactive with peanut lectin suggesting that RCCT cells are a mixture of principal and intercalated cells. Approximately 10(7) RCCT cells were obtained after 4 to 5 days in primary culture. Moreover, RCCT cells continued to proliferate after passaging with a doubling time of approximately 32 h. RCCT cells passaged once and then cultured 4-5 days were found 1) to synthesize cAMP in response to arginine vasopressin (AVP), prostaglandin E2 (PGE2), isoproterenol, and parathyroid hormone, but not calcitonin, prostaglandin D2, or prostaglandin I, and 2) to release PGE2 in response to bradykinin but not arginine vasopressin or isoproterenol. Our results indicate that cultured RCCT cells retain many of the hormonal, histochemical, and morphological properties expected for a mixture of principal and intercalated rabbit cortical collecting tubule epithelia. RCCT cells should prove useful both for studying hormonal interactions in the cortical collecting tubule and as a starting population for isolating intercalated collecting tubule epithelia.

Identification of inhibitory and calmodulin-binding domains of the PDE1A1 and PDE1A2 calmodulin-stimulated cyclic nucleotide phosphodiesterases.

Using a bovine 61-kDa (PDE1A2) calmodulin-stimulated phosphodiesterase (CaM-PDE) cDNA and a bovine lung 59-kDa (PDE1A1) CaM-PDE cDNA reported here, we have identified two new regions within the primary structure of these two related isozymes that are important for regulation by Ca2+/CaM. PDE1A1 is identical to the PDE1A2 isozyme except for the amino-terminal 18 residues. In agreement with earlier studies, the CaM concentration required for half-maximal activation (KCaM) of recombinant PDE1A1 (0.3 nM) was approximately 10-fold less than the KCaM for recombinant PDE1A2 (4 nM). A series of deletion mutations of the PDE1A2 cDNA removing nucleotide sequence encoding the first 46-106 aminoterminal residues were constructed and expressed using the baculovirus system. Deletion of the amino acids encompassing a previously identified, putative CaM-binding domain (residues 4-46) produced a polypeptide that was still activated 3-fold by CaM (KCaM approximately 3 nM). However, complete CaM-independent activation occurred when residues 4-98 were deleted. To determine the location of the additional CaM-binding domain(s), the inhibitory potency of seven overlapping, synthetic peptides spanning amino acids 76-149 of PDE1A2 was tested using the CaM-activated enzyme. One peptide spanning amino acids 114-137 of PDE1A2 appeared to be the most potent inhibitor of CaM-stimulated activity. These results reveal the existence of a CaM-binding domain located approximately 90 residues carboxyl-terminal to the putative CaM-binding domains previously identified within the PDE1A1 and PDE1A2 isozymes. Moreover, a discrete segment important for holding these CaM-PDEs in a less active state at low Ca2+ concentrations is located between the two CaM-binding domains.

Identification, quantitation, and cellular localization of PDE1 calmodulin-stimulated cyclic nucleotide phosphodiesterases.

The calmodulin-stimulated cyclic nucleotide phosphodiesterases (PDE1s) constitute a large gene family and are found in a wide variety of tissues and cells. Because of the functional diversity of PDE1 genes and the observation that these isozymes often make up a major component of the total cyclic nucleotide hydrolytic activity in certain cell types, PDE1s are of growing interest as targets for therapeutic intervention. Here we describe a series of methodologies to identify, quantitate, and determine the cellular expression of PDE1 isozymes. We describe first the resolution of different PDEs using high-performance anion-exchange chromatography and then a Western blotting methodology for identifying or authenticating PDE1 activities. Next we present an immunoprecipitation method that can be used for quantitating specific PDE1 isoforms and describe the use of RNase protection analysis for further identification of PDE1 subtypes. Finally, we provide a simple, immunocytochemical method for determining the cellular expression of PDE1 isozymes. Combined, the above methodologies should allow an investigator to identify, quantitate, and determine the cellular localization of PDE1 isozymes in any tissue with little ambiguity.