Subcellular localizations of guanylate cyclase and 3',5'-cyclic nucleotide phosphodiesterase in sea urchin sperm.

The subcellular localizations of guanylate cyclase and 3',5'-cyclic nucleotide phosphodiesterase in sea urchin sperm were examined. Both the specific and total activities of these two enzymes were much higher in sperm flagella (tails) than in the heads. In addition to the observation that guanylate cyclase in the flagella was particulate-bound and solubilized by Triton X-100, more than 80% of the cyclase activity in the flagella was found in the plasma membrane fraction, whereas the activity of cyclic nucleotide phosphodiesterase was observed in both the axonemal and plasma membrane fractions. The observations indicated that the cyclase in the flagella appeared to be associated with the plasma membrane. Cyclic nucleotide phosphodiesterase in the plasma membrane fraction as well as the axonemal fraction hydrolyzed both cyclic GMP and cyclic AMP; however, the rates of hydrolysis for cyclic GMP were obviously higher than those for cyclic AMP. The enzymic properties of guanylate cyclase and cyclic nucleotide phosphodiesterase in sperm flagella were also briefly described.     

Subcellualr localizations of guanylate cyclase and 3′,5′-cyclic nucleotide phophodiesterase in sea urchin sperm

The subcellular localizations of guanylate cyclase and 3′,5′-cyclic nucleotide phophodiesterase in sea urchin sperm were examined. Both the specific and total activities of these two enzymes were much higher in sperm flagella (tails) than in the heads. In addition to the observation that guanylate cyclase in the flagella was particulate-bound and solubilized by Triton X-100, more than 980% of the cyclase activity in the flagella was found in the plasma membrane fraction, whereas the activity of cyclic nucleotide phosphodiesterase was observed in both the axonemal and plasma membrane fractions. The observations indicated that the cyclase in the flagella appeared to be associated with the plasma membrane. Cyclic nucleotide phosphodiesterase in the plasma membrane fraction as well as the axonemal fraction hydrolyzed both cyclic GMP and cyclic AMP; however, the rates of hydrolysis for cyclic GMP were obviously higher than those for cyclic AMP. The enzymic properties of guanylate cyclase and cyclic nucelotide phosphodiesterase in sperm flagella were also briefly described.     

Regional differentiation of the sea urchin sperm plasma membrane

In order to study the molecular basis for the functional localization and behavioral control of sperm, we have partially characterized plasma membranes prepared from isolated head and tail fractions. These membranes have similar amounts of the Na+ pump (as reflected by (Na+,K+)-ATPase activity), whereas they differ in protein composition, binding sites for Ca2+ channel antagonists, and in the localization of enzymes of cyclic nucleotide metabolism. The Ca2+ channel antagonist D600 (and related phenylalkylamines) binds to plasma membrane preparations from sperm heads and tails with much higher affinity than do the dihydropyridine antagonists. This binding is inhibited greatly by certain monovalent (but not divalent) ions, especially Na+, Tris+, glycine ethyl ester+, and methylamine+.K+,Li+, and choline+ are less effective. In media of ionic composition resembling seawater, sperm tail membranes exhibit 6.5-fold more binding sites for D600 than do membranes from sperm head. cGMP phosphodiesterase and adenylate cyclase are also enriched in plasma membranes from the tail. Thus, the highly polarized sperm cell exhibits a regional differentiation of plasma membrane proteins implicated in behavioral control.     

Cyclic nucleotides and platelet aggregation. Effect of aggregating agents on the activity of cyclic nucleotide-metabolizing enzymes.

The activities of adenylate and guanylate cyclase and cyclic nucleotide 3':5'-phosphodiesterase were determined during the aggregation of human blood platelets with thrombin, ADP, arachidonic acid and epinephrine. The activity of guanylate cyclase is altered to a much larger degree than adenylate cyclase, while cyclic nucleotide phosphodiesterease activity remains unchanged. During the early phases of thrombin-and ADP-induced platelet aggregation a marked activation of the guanylate cyclase occurs whereas aggregation induced by arachidonic acid or epinephrine results in a rapid diminution of this activity. In all four cases, the adenylate cyclase activity is only slightly decreased when examined under identical conditions. Platelet aggregation induced by a wide variety of aggregating agents including collagen and platelet isoantibodies results in the "release" of only small amounts (1-3%) of guanylate cyclase and cyclic nucleotide phosphodiesterase and no adenylate cyclase. The guanylate cyclase and cyclic nucleotide phosphodiesterase activities are associated almost entirely with the soluble cytoplasmic fraction of the platelet, while the adenylate cyclase if found exclusively in a membrane bound form. ADP and epinephrine moderately inhibit guanylate and adenylate cyclase in subcellular preparations, while arachidonic and other unsaturated fatty acids moderately stimulate (2-4-fold) the former. It is concluded that (1) the activity of platelet guanylate cyclase during aggregation depends on the nature and mode of action of the inducing agent, (2) the activity of the membrnae adenylate cyclase during aggregation is independent of the aggregating agent and is associated with a reduction of activity and (3) cyclic nucleotide phosphodiesterase remains unchanged during the process of platelet aggregation and release. Furthermore, these observations suggest a role for unsaturated fatty acids in the control of intracellular cyclic GMP levels.     

Translocation of phospholipid/Ca2+-dependent protein kinase in B-lymphocytes activated by phorbol ester or cross-linking of membrane immunoglobulin.

Treatment of hepatocytes with islet activating protein (pertussis toxin) from Bordetella pertussis blocked the ability of insulin to inhibit adenylate cyclase activity both in broken plasma membranes and in intact hepatocytes. Such treatment of intact hepatocytes with pertussis toxin did not prevent insulin from activating the peripheral plasma membrane cyclic AMP phosphodiesterase although it did inhibit the ability of insulin to activate the 'dense-vesicle' cyclic AMP phosphodiesterase. The ability of glucagon pretreatment of hepatocytes to block insulin's activation of the plasma membrane cyclic AMP phosphodiesterase was abolished in pertussis toxin-treated hepatocytes. It is suggested that the ability of insulin to manipulate cyclic AMP concentrations by inhibiting adenylate cyclase and activating the plasma membrane and 'dense-vesicle' cyclic AMP phosphodiesterases involves interactions with the guanine nucleotide regulatory protein system occurring in liver plasma membranes.     

Gossypol modulation of nucleotide metabolizing enzymes in the reproductive tract of male rats

The activities of several pivotal nucleotide metabolizing enzymes from the testis and vasal sperm of rats treated for 7 wk with 0, 20 or 30 mg X kg X day gossypol acetic acid were examined. Total testicular lactate dehydrogenase (LDH) activity increased 40% above control in the highest treatment group examined. However, the specific activity of the testis-specific isozyme of LDH, LDH-C4, decreased to 50 and 20% of control in the 20 and 30 mg X kg X day treatment groups, respectively. Basal soluble adenylate cyclase from a 100,000 X g supernatant of testis homogenate exhibited a 25% decrease in activity only in the 30-mg treatment group. Basal adenylate cyclase activity in the testicular membrane fraction increased 20 to 30% above control in response to gossypol administration. Testis membranes from the 20- and 30-mg treatment group exhibited a 2- and 4-fold greater activation of adenylate cyclase by guanine nucleotides. In vitro dose-response curves showed a half-maximal inhibitory concentration (IC50) for inhibition of soluble testicular adenylate cyclase by gossypol of 400 microM in each treatment group. Caudal epididymal sperm adenylate cyclase activity decreased to 25% of control levels in gossypol-treated animals, and the in vitro sensitivity of the enzyme to the inhibitory effects of gossypol increased 4-fold. IC50 values for gossypol inhibition of sperm adenylate cyclase decreased from 200 microM in control animals to 75 and 50 microM in the 20 and 30 mg X kg X day treatment groups, respectively. Cyclic adenosine 3':5' monophosphate phosphodiesterase activity in caudal sperm increased 6-fold in the 20- and 30-mg treatment groups. These results demonstrate that nucleotide metabolizing enzymes in sperm are major targets for the actions of gossypol and provide a possible mechanism for the inhibition of normal sperm function by this compound.     

Adenylate cyclase and cyclic AMP phosphodiesterase in Bradyrhizobium japonicum bacteroids.

Adenylate cyclase and cyclic AMP (cAMP) phosphodiesterase have been identified and partially characterized in bacteroids of Bradyrhizobium japonicum 3I1b-143. Adenylate cyclase activity was found in the bacteroid membrane fraction, whereas cAMP phosphodiesterase activity was located in both the membrane and the cytosol. In contrast to other microorganisms, B. japonicum adenylate cyclase remained firmly bound to the membrane during treatment with detergents. Adenylate cyclase was activated four- to fivefold by 0.01% sodium dodecyl sulfate (SDS), whereas other detergents gave only slight activation. SDS had no effect on the membrane-bound cAMP phosphodiesterase but strongly inhibited the soluble enzyme, indicating that the two enzymes are different. All three enzymes were characterized by their kinetic constants, pH optima, and divalent metal ion requirements. With increasing nodule age, adenylate cyclase activity increased, the membrane-bound cAMP phosphodiesterase decreased, and the soluble cAMP phosphodiesterase remained largely unchanged. These results suggest that cAMP plays a role in symbiosis.     

Rat sperm enzymes during epididymal transit

The activities of cAMP and cGMP phosphodiesterases (EC 3.1.4.1), adenylate cyclase (EC 4.6.1.1) and protein carboxyl-methylase (EC 2.1.1.24) were measured in the particulate and soluble (105 000 g supernatant) fractions of washed spermatozoa isolated from five segments of the adult rat epididymis. The activities of both phosphodiesterases decreased during epididymal transit, whereas adenylate cyclase and protein carboxyl-methylase underwent a progressive increase, the latter showing the most marked alteration. Both cAMP and cGMP phosphodiesterases as well as the adenylate cyclase were all associated primarily with the particulate fraction, and the extent to which these enzymes were associated with the membranes increased as the spermatozoa passed through the epididymis. Sperm protein carboxyl-methylase activity was, on the other hand, predominantly soluble in all segments of the epididymis. Adenylate cyclase, cAMP phosphodiesterase and protein carboxyl-methylase activities were found predominantly in the sperm tails, whereas cGMP phosphodiesterase was equally distributed between heads and tails. These observations imply that the acknowledged increase in intracellular cAMP levels which occurs in spermatozoa during epididymal transit may be a consequence of both increased synthesis (adenylate cyclase) and reduced hydrolysis (phosphodiesterase).     

Ultracytochemical localizations of adenylate cyclase, guanylate cyclase and cyclic 3',5'-nucleotide phosphodiesterase activity on the trophoblast in the human placenta. Direct histochemical evidence

Ultracytochemical localizations of cyclic nucleotide-metabolizing enzymes, namely adenylate cyclase (AC), guanylate cyclase (GC) and cyclic 3',5'-nucleotide phosphodiesterase (PDE), have been demonstrated in the human term placenta. AC activity was found positive on the basal plasma membrane of the syncytiotrophoblast and on the pinocytotic vesicle of the fetal capillary endothelial cell. GC activity was observed to be strong on the plasma membrane of the microvilli of the syncytiotrophoblast. The cAMP PDE activity was shown positive both on the basal plasma membrane and on the microvillous membrane, while cGMP PDE activity was exclusively confined to the microvilli of the syncytiotrophoblast. These observations suggest that the syncytiotrophoblast plays an important role in the cyclic nucleotide metabolism in the human term placenta and that there might be significant functional differences between its basal plasma membrane and its microvillous membrane.     

Short-term changes in levels of cyclic AMP, adenylate cyclase, and phosphodiesterase during the initiation of sperm motility in rainbow trout

In order to clarify the role of the system that generates and degrades cyclic AMP during the initiation of motility of trout sperm, short-term changes in levels of intraspermatozoal cyclic AMP, adenylate cyclase, and phosphodiesterase were measured. Levels of cyclic AMP and the activity of adenylate cyclase increased and reached a maximum level 1 sec after transfer of sperm to K+-free medium, where they became motile, and then decreased rapidly. However, there were no changes in either parameter in sperm which remained immotile in K+-rich medium. In addition, an increase in the activity of phosphodiesterase was observed 4 sec later than the increase in levels of cyclic AMP and adenylate cyclase. These findings suggest that a very rapid change in the level of intracellular cyclic AMP occurs within 1 sec, at the moment of spawning, by the activation of adenylate cyclase and phosphodiesterase, and regulates the initiation of trout sperm motility.     

Simulations of the roles of multiple cyclic nucleotide phosphodiesterases.

1. Simulations were performed using a model for cellular cyclic AMP metabolism involving a hormone-activated adenylate cyclase and two cyclic nucleotide phosphodiesterases with different Michaelis constants. 2. The response curves of cyclic AMP concentration as a function of hormone concentration were affected by regulating the phosphodiesterases. The maximum velocity of the high-affinity phosphodiesterase (V1) was important in determining the position of the response curve; when v1 was less than the maximal activity of adenylate cyclase (Vc), sigmoid response curves were readily produced. The maximum attainable concentration of cyclic AMP was determined primarily by V1 when Vc less than V1, and primarily by the activity of the low-affinity enzyme when Vc greater than V1 (V2 much greater than Vc in all cases). 3. The glucagon-stimulated adenylate cyclase and insulin-stimulated phosphodiesterase of the rat liver plasma membrane were simulated using experimentally determined values for the enzyme-kinetic parameters, and a considerable potential for regulation of the system by insulin was demonstrated. 4. Other possible functions for the regulation of phosphodiesterases are considered, in particular the value of increasing the speed of response to decreases in hormone concentration.     

Sea urchin sperm guanylate cyclase antibody. Cross-reactivity various rat tissue guanylate cyclases.

After the repeated injection of sea urchin sperm guanylate cyclase into rabbits, antibodies to the enzyme were formed. These antibodies inhibited the particulate or the Triton-dispersed forms of the sperm enzyme by greater than 97%. The sperm adenylate cyclase, cyclic GMP phosphodiesterase, adenosine triphosphatase, guanosine triphosphatase, and 5'-nucleotidase enzymes were not affected by the antiserum. The antiserum inhibited the Triton-dispersed guanylate cyclase from rat heart, liver, lung, spleen, and kidney but did not inhibit the soluble form of the enzyme from any of these tissues. The inhibition of the Triton-dispersed enzyme in these tissues was partial, however, ranging from 30% (liver) to 70% (heart). These results provide evidence that adenylate cyclase is antigenically different from guanylate cyclase, and that the soluble form of guanylate cyclase is antigenically different from a particulate form of the enzyme in various rat tissues.     

The phorbol ester TPA prevents the expression of both glucagon desensitisation and the glucagon-mediated block of insulin stimulation of the peripheral plasma membrane cyclic AMP phosphodiesterase in rat hepatocytes

The phorbol ester TPA (12-O-tetradecanoyl phorbol-13-acetate) causes a dose-dependent inhibition of the glucagon-stimulated adenylate cyclase activity expressed in plasma membranes isolated from TPA-treated hepatocytes. However, no observable inhibitory effect of TPA on adenylate cyclase activity was observed in cells which had been exposed to glucagon for 5 min, prior to isolation, to desensitise adenylate cyclase. The degree of inhibition of adenylate cyclase elicited by both glucagon desensitisation and TPA treatment of hepatocytes was identical. Pre-treatment of hepatocytes with TPA was also found to prevent glucagon from blocking insulin's activation of the peripheral plasma membrane cyclic AMP phosphodiesterase in intact hepatocytes. TPA treatment also inhibited the ability of cholera toxin to activate the peripheral cyclic AMP phosphodiesterase in intact hepatocytes. It is suggested that in these particular instances TPA and glucagon elicit mutually exclusive processes rather than TPA mimicking glucagon desensitisation per se.     

Activation of spermatozoan adenylate cyclase by a low molecular weight factor in porcine seminal plasma

The ejaculated porcine spermatozoa were fractionated into the cytosol, membrane, midpiece plus tail (flagella) and head fractions, and their adenylate cyclase activities were measured. About 65% of the total activity was located in the flagella fraction. For all the fractions, Mn2+-dependent adenylate cyclase activity was about 20 times higher than Mg2+-dependent activity, and guanine nucleotides, fluoride, and other reagents tested did not activate adenylate cyclase. The results suggest that the GTP-dependent regulatory subunit is absent in porcine spermatozoa. The porcine seminal plasma was found to stimulate the adenylate cyclase activity in spermatozoa. The stimulating factor in porcine seminal plasma was partially purified by gel filtration and the molecular weight of the factor appeared to be between 200 and 300. The partially purified factor is heat stable and is not inactivated by treatment with Pronase, trypsin, phospholipase A2 or D but is inactivated by acid hydrolysis. It is easily soluble in water, partially soluble in methanol, and insoluble in ethanol, ethyl ether, chloroform, or acetone. The activation of sperm adenylate cyclase by the factor occurred without a time lag. The activating effect was dose-dependent, saturated at high dose, and ascribed to the increase of the maximal velocity (Vmax). The effect of the factor appears to be limited to adenylate cyclase in spermatozoa; the factor activated adenylate cyclase both in porcine and bovine spermatozoa but failed to activate those in other porcine tissues. The factor was shown to activate the enzyme not only in the ejaculated spermatozoa but also in the epididymal sperm. The factor was also found to elevate the cAMP level in the intact porcine spermatozoa. The factor enhanced the motility of corpus and cauda epididymal spermatozoa. These findings indicate the possibility that this factor initiates the spermatozoan motility upon ejaculation through directly activating adenylate cyclase.     

The subcellular localization of calmodulin, cyclic AMP phosphodiesterase, and adenylate cyclase in bovine adrenal medulla

The subcellular localization of calmodulin, cyclic nucleotide phosphodiesterase, and adenylate cyclase was studied in bovine adrenal medulla. Approximately 70% of the calmodulin and 90% of the cAMP phosphodiesterase activities were found colocalized in the cytoplasm. The subcellular distribution of adenylate cyclase closely paralleled the distribution of acetylcholinesterase, a marker for plasma membranes. The fraction of calmodulin which is particulate in nature has a distribution profile very similar to that of adenylate cyclase. The chromaffin granule fraction contained only 0.86% of the total cAMP phosphodiesterase, 0.41% of the total adenylate cyclase, and 1.4% of the total calmodulin.     

Histidine regulation of cyclic AMP metabolism in cultured renal epithelial LLC-PK1 cells.

L-Histidine and imidazole (the histidine side chain) significantly increase cAMP accumulation in intact LLC-PK1 cells. This effect is completely inhibited by isobutylmethylxanthine (IBMX). Histidine and imidazole stimulate cAMP phosphodiesterase activity in soluble and membrane fractions of LLC-PK1 cells suggesting that the IBMX-sensitive effect of these agents to stimulate cAMP formation is not due to inhibition of cAMP phosphodiesterase. Histidine and imidazole but not alanine (the histidine core structure) increase basal, GTP-, forskolin-, and AVP-stimulated adenylate cyclase activity in LLC-PK1 membranes. Two other amino acids with charged side chains (aspartic and glutamic acids) increase AVP-stimulated but neither basal- nor forskolin-stimulated adenylate cyclase activity. This suggests that multiple amino acids with charged side chains can regulate selected aspects of adenylate cyclase activity. To better define the mechanism of histidine regulation of adenylate cyclase, membranes were detergent-solubilized which prevents histidine and imidazole potentiation of forskolin-stimulated adenylate cyclase activity and suggests that an intact plasma membrane environment is required for potentiation. Neither pertussis toxin nor indomethacin pretreatment alter imidazole potentiation of adenylate cyclase. IBMX pretreatment of LLC-PK1 membranes also prevents imidazole to potentiate adenylate cyclase activity. Since IBMX inhibits adenylate cyclase coupled adenosine receptors, LLC-PK1 cells were incubated in vitro with 5'-N-ethylcarboxyamideadenosine (NECA) which produced a homologous pattern of desensitization of NECA to stimulate adenylate cyclase activity. Despite homologous desensitization, histidine and imidazole potentiation of adenylate cyclase was unaltered. These data suggest that histidine, acting via an imidazole ring, potentiates adenylate cyclase activity and thereby increases cAMP formation in cultured LLC-PK1 epithelial cells. This potentiation requires an intact plasma membrane environment, occurs independent of a pertussis toxin-sensitive substrate and of products of cyclooxygenase, and is inhibited by IBMX. This IBMX-sensitive pathway does not involve either inhibition of cAMP phosphodiesterase activity or a stimulatory adenosine receptor coupled to adenylate cyclase.     

Evidence that the mitochondrial activator of phosphorylated branched-chain 2-oxoacid dehydrogenase complex is the dissociated E1 component of the complex

The ability of glucagon (10 nM) to increase hepatocyte intracellular cyclic AMP concentrations was reduced markedly by the tumour-promoting phorbol ester TPA (12-O-tetradecanoyl phorbol-13-acetate). The half-maximal inhibitory effect occurred at 0.14 ng/ml TPA. This action occurred in the presence of the cyclic AMP phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) indicating that TPA inhibited glucagon-stimulated adenylate cyclase activity. TPA did not affect either the binding of glucagon to its receptor or ATP concentrations within the cell. TPA did inhibit the increase in intracellular cyclic AMP initiated by the action of cholera toxin (1 microgram/ml) under conditions where phosphodiesterase activity was blocked. TPA did not inhibit glucagon-stimulated adenylate cyclase activity in a broken plasma membrane preparation unless Ca2+, phosphatidylserine and ATP were also present. It is suggested that TPA exerts its inhibitory effect on adenylate cyclase through the action of protein kinase C. This action is presumed to be exerted at the point of regulation of adenylate cyclase by guanine nucleotides.     

Investigations on adenosine 3',5'-monophosphate phosphodiesterase in ram semen and initial characterization of a sperm-specific isoenzyme.

Phosphodiesterase is shown to occur in ram semen, and its activity to be higher in spermatozoa than in seminal plasma. Using similar substrate levels, the rate at which adenosine 3',5'-monophosphate (cyclic AMP) is metabolized by phosphodiesterase in spermatozoa is about 100 times higher than that of cyclic AMP synthesis by adenylate cyclase. In spermatozoa, phosphodiesterase is present partly in a soluble form, and partly bound; both forms can be extracted by sonication. The soluble enzyme (pH optimum 8-0, Km = 1-5 muM, mol. wt 165,000) occurs as a single isoenzyme, as shown by polyacrylamide gel electrophoresis and anion-exchange chromatography; this isoenzyme appears to be specific for spermatozoa and its formation in the testis coincides with the appearance of spermatozoa. The bound sperm enzyme has been solubilized with Trion X-100; it is a single isoenzyme (pH optimum 8-0, mol. wt 165,000) which is electrophoretically different from the soluble form, but similar to the phosphodiesterase found in other tissues. Seminal plasma phosphodiesterase (pH optimum 8-8, mol. wt 165,000) is present in the form of three isoenzymes; all three are different from the two forms of sperm phosphodiesterase, but are similar to the isoenzymes found in certain male accessory organs.     

Adenine nucleotide changes at initiation of bull sperm motility.

Testicular and cauda epididymal sperm were obtained via catheters previously implanted in the rete testis and proximal vas deferens of bulls and were used to examine the relationships among sperm motility, cyclic adenosine 3':5'-monophosphate (cAMP) level, adenine nucleotide levels, and rates of glucose and oxygen consumption. Testicular, cauda epididymal, and ejaculated sperm contain cAMP-stimulated protein kinase, adenylate cyclase, and nucleotide phosphodiesterase. Treatment of the nonmotile testicular sperm with phosphodiesterase inhibitors resulted in a doubling of cellular cAMP concentration and a 25% increase in their glucose consumption. No change in motility, ATP level, or rate of oxygen consumption was observed. Sperm in neat cauda epididymal semen had flagellating tails but no progressive motility. Dilution of these sperm into glucose-containing buffer resulted in an increase in intracellular cAMP concentration and a decrease in ATP level with concomitant increases in ADP and AMP levels. These biochemical changes occurred within 30 s after dilution and apparently preceded the initiation of progressive motility by most cells. Since sperm in neat cauda epididymal semen became progressively motile when diluted with neat cauda epididymal plasma as well as accessory sex gland fluid or buffer, composition of the fluid surrounding the sperm is not responsible for the initiation of progressive motility upon dilution nor does cauda epididymal plasma contain an inhibitory factor. Perhaps release from contact immobilization provides the stimulation for the initial acquisition of progressive motility by cauda epididymal sperm. We conclude that during epididymal passage sperm develop from a cell physically unresponsive to changes in cAMP concentration to a form which initiates progressive motility upon changes in cAMP concentration.     

Distribution of parathyroid hormone-stimulated adenylate cyclase in plasma membranes of cells of the kidney cortex.

Free flow electrophoresis was employed to separate renal cortical plasma membranes into luminal (brush border microvilli) and contraluminal (basal-lateral membrane) fractions. During the separation adenylate cyclase activity was found to parallel the activity of Na+-K+-activated ATPase, an enzyme which is present in contraluminal but not in luminal membranes. In the basal-lateral membrane fraction the specific activities of adenylate cyclase and Na+-K+-activated ATPase were 4.4 and 4.6 times greater, respectively, than in the brush border fraction. The adenylate cyclase of the basal-lateral membrane fraction was specifically stimulated by parathyroid hormone which maximally increased enzyme activity eightfold. The biologically active (1-34) peptide fragment of paratyhroid hormone produced a 350% increase in adenylate cyclase activity. In contrast, calcitonin, epinephrine and vasopressin maximally stimulated the enzyme by only 55, 35 and 30%, respectively. These results indicate that adenylate cyclase, specifically stimulated by parathyroid hormone, is distributed preferentially in the contraluminal region of the plasma membrane of renal cortical epithelial cells.