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F R DeRubertis

Publications and source records attributed to F R DeRubertis.

At least 73 records · Page 4Linked to original sources
PubMed1984

Bile salt stimulation of colonic epithelial proliferation. Evidence for involvement of lipoxygenase products.

Prostaglandin E2 (PGE2) and several other prostaglandins synthesized by colon suppress the proliferative activity of colonic epithelium. However, bile salts stimulate colonic epithelial proliferation despite the actions of bile salts to enhance the release of arachidonate and consequent colonic synthesis of PGE2. The current study was conducted to assess whether bile salt-induced increases in colonic formation of arachidonate metabolites other than PGE2 were linked to the stimulation of the proliferative activity of colonic epithelium. Within 10 min of addition, deoxycholate markedly stimulated the in vitro release of [14C]arachidonate from prelabeled rat colon. When given in vivo by intracolonic instillation deoxycholate (10 mumol) increased colonic accumulation of immunoreactive prostaglandin E (PGE), thromboxane B2 (TXB2), and the lipoxygenase product 12-hydroxyeicosatetraenoic acid (12-HETE) by two to fourfold over control in 30 min. This effect of intracolonic deoxycholate was followed by a ninefold increase in mucosal ornithine decarboxylase activity (4 h), and a subsequent two to threefold increase in [3H]thymidine [( 3H]Thd) incorporation into DNA of either mucosal scrapings or isolated pools of proliferative colonic epithelial cells (24 h). Intracolonic instillation of indomethacin (50 mumol) suppressed to low or undetectable levels both basal colonic accumulation of PGE and TXB2 and the increases in each parameter induced by subsequent instillation of deoxycholate. By contrast, indomethacin enhanced accumulation of 12-HETE in both control colons and those subsequently exposed to deoxycholate. The increases in 12-HETE induced by indomethacin alone were correlated with stimulation of mucosal ornithine decarboxylase activity and [3H]Thd incorporation into mucosal DNA. Indomethacin also enhanced the increases in these parameters induced by deoxycholate. Intracolonic instillation of phenidone (25-100 mumol) suppressed accumulation of PGE, TXB2, and 12-HETE in control colons and the increases in these parameters induced by a subsequent instillation of deoxycholate. Phenidone alone did not alter mucosal ornithine decarboxylase activity or [3H]thymidine incorporation into mucosal DNA. However, phenidone suppressed or abolished increases in these parameters induced by a subsequent instillation of deoxycholate. 4-(2-[IH-imidazol-1-yl]ethoxy) benzoic acid hydrochloride UK 37,248, which selectively reduced colonic TXB2 to undetectable levels without altering PGE or 12-HETE, had no effect on control or deoxycholate-induced increases in mucosal ornithine decarboxylase activity or [3H]Thd incorporation into DNA. Neither indomethacin nor phenidone altered the increases in [(14)C]arachidonate release induced in vitro by deoxycholate. Chenodeoxycholate and cholate also stimulated [(14)C]arachidonate release from colon in vitro within 10 min, and increased colonic 12-HETE (30 min) and mucosal ornithine decarboxylase activity (4 h) upon intracolonic installation. Prior installation of phenidone inhibited the increases in both 12-HETE and ornithine decarboxylase activity induced by these bile salts. The results support a role for bile salt-induced increases in colonic accumulation of lipoxygenase products, as reflected by 12-HETE, in the subsequent stimulation of the proliferative activity of colonic epithelium.

Animals↗
PubMed1983

Requirement for heme in the activation of purified guanylate cyclase by nitric oxide.

Guanylate cyclase activity was purified to apparent homogeneity from rat liver (7700-fold) and bovine lung (8600-fold) soluble fractions by ammonium sulfate precipitation, DEAE-cellulose chromatography, agarose gel filtration and isoelectric focussing. The purified enzymes did not contain heme and did not respond to NO, nitroprusside or NO-cysteine in the absence of exogenous hematin. By contrast, preformed NO-hemoglobin increased enzyme activity 10-12-fold or 60-80-fold when 4 mM MnCl2 or 4 mM MgCl2, respectively, were employed as the metal ion co-factor. Addition of hematin to the enzyme preparations restored responsiveness to NO, nitroprusside or NO-cysteine to levels seen with NO-hemoglobin. Partial purification of guanylate cyclase from the soluble fraction of bovine lung (2400-fold) by ammonium sulfate precipitation, DEAE-cellulose chromatography, agarose gel filtration and high pressure liquid chromatography (HPLC) resulted in a preparation which contained endogenous heme as indicated by absorbance at 436 nm and responded to NO, nitroprusside and NO-cysteine in the absence of added hematin. By contrast, guanylate cyclase purified from the hepatic supernatant by the identical procedure, did not contain detectable absorption due to heme and did not respond or responded poorly to NO, nitroprusside or NO-cysteine in the absence of exogenous hematin. Analogous to hepatic guanylate cyclase purified by isoelectric focussing, the HPLC purified hepatic enzyme was activated 14-fold by NO-hemoglobin in assays which contained 4 mM MnCl2 and 60-fold in assays with 4 mM MgCl2. Further, addition of hematin to the HPLC purified enzyme restored responsiveness to NO, nitroprusside and NO-cysteine to levels seen with NO-hemoglobin. These effects of hematin were specific for hematin and were not mimicked by albumin, sucrose or dithiothreitol. Moreover, the failure to observe stimulation of purified hepatic guanylate cyclase was not explained by a shift in the concentration response relationship between NO and guanylate cyclase activity. Several observations indicated that neither NO-thiol complexes nor [Fe(CN)5NO]-3 were the proximate moieties responsible for activation of guanylate cyclase by nitroprusside and related agents, as has been previously suggested. These results strongly support the proposal that activation of guanylate cyclase by NO and related agents specifically requires formation of an NO-heme complex.

Anaerobiosis↗
PubMed1983

Properties of multiple kinetic forms of soluble cyclic nucleotide phosphodiesterase activity of rat colonic mucosa.

Soluble phosphodiesterase (EC 3.1.4.1) activity is 3-5-fold lower in superficial colonic epithelial cells compared to that in cells isolated from the lower colonic crypt. Higher phosphodiesterase activity in lower crypt cells is correlated with a 5-fold higher rate of incorporation of [3H]thymidine into DNA in these cells. DEAE-cellulose chromatography of the soluble fraction of superficial and proliferative colonic epithelial cells resulted in separation of three enzyme forms: (1) fraction I, an enzyme which hydrolyzes both cAMP and cGMP with high affinity (apparent Km cAMP = 5 +/- 1 microM, Km cGMP = 2.5 +/- 0.5 microM) and is stimulated 3-6-fold by Ca2+ plus calmodulin; (2) fraction II, a form which hydrolyzes both cAMP and cGMP with low affinity (S0.5 cAMP = 52 +/- 7 microM, S0.5 cGMP = 17 +/- 4 microM), exhibits positive copperativity with respect to substrate and shows cGMP stimulation of cAMP hydrolysis and (3) fraction III, a cAMP-specific form which exhibits biphasic kinetics, a low Km for cAMP (Km cAMP = 5 +/- 1 microM) and does not hydrolyze cGMP. The pattern of distribution of phosphodiesterase activities on DEAE-cellulose was similar in superficial and proliferative colonic epithelial cells. The higher specific activity in proliferative cells was reflected in higher activities of each of the three chromatographically distinct forms of the enzyme. In contrast to epithelial cells, the soluble fraction of homogenates of the submucosa and supporting cells exhibited phosphodiesterase forms I and II and was lacking in the form corresponding to fraction III of epithelial cells.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1983

Ca2+.Calmodulin-dependent release of arachidonic acid for renal medullary prostaglandin synthesis. Evidence for involvement of phospholipases A2 and C.

The present study examined (a) the source of arachidonic acid for Ca2+-stimulated renal inner medullary prostaglandin synthesis, (b) the Ca2+-dependence of enzymes of the phospholipase A2 and C pathways, and (c) the role of calmodulin in these Ca2+ actions. Ca2+ plus the ionophore A23187 stimulated (2-4-fold) release of labeled arachidonate, diglyceride, prostaglandin E2 or F2 alpha from inner medullary slices with a concomitant fall in labeled phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide hydrochloride (W-7) (10-100 microM) abolished or suppressed Ca++-stimulated immunoreactive prostaglandin E, labeled arachidonate and prostaglandin release, and the fall in labeled phospholipids but did not suppress labeled diglyceride or inositol accumulation. Studies in subcellular fractions demonstrated a particulate phospholipase A2 activity and a phosphatidylinositol-specific phospholipase C activity which was predominantly soluble (80%). W-7 or trifluoperazine (25 microM) abolished Ca2+-stimulated phospholipase A2 activity and particulate phospholipase C activity but were without effect on soluble phospholipase C. W-7 (100 microM) was without effect on Ca2+-stimulated diglyceride lipase and phosphatidic acid-specific phospholipase A2 activities. Hypertonic urea at concentrations that pertain in the inner medulla of hydropenic rats in vivo inhibited Ca2+-induced increases in labeled arachidonate release and immunoreactive prostaglandin E in slice incubates and Ca2+-responsive phospholipase C and A2. The results are consistent with the involvement of phospholipase A2, C, or both in the Ca2+ (+A23187)-stimulated release of free arachidonate for prostaglandin synthesis and support a role for calmodulin in Ca2+ activation of phospholipase A2 and particulate phospholipase C.

Animals↗
PubMed1983

Patterns of prostaglandin synthesis and degradation in isolated superficial and proliferative colonic epithelial cells compared to residual colon.

Prostaglandin (PG) synthesis and degradation were examined in different regions (epithelial versus non-epithelial structures) of the rat distal colon by both HPLC analysis of [14C] arachidonate (AA) metabolites and by specific radioimmunoassays. Intact isolated colonic epithelial cells synthesized mainly PGF2 alpha and TXA2, as monitored from the formation of its stable degradation product TXB2 (PGF2 alpha greater than TXB2 greater than 6-keto-PGF1 alpha, the stable degradation product of PGI2 = PGD2 = PGE2 = 13,14-dihydro-15-keto-PGF2 alpha). The profile of PG products of isolated surface epithelial cells was identical to that of proliferative epithelial cells. However, generation of PGs by surface epithelium was 2 to 3-fold higher than by proliferative cells both basally and in the presence of a maximal stimulating concentration (0.1 mM) of AA. The latter implied a greater synthetic capacity of surface epithelium, rather than differences due to endogenous AA availability. The major sites of PG synthesis in colon clearly resided in submucosal structures; the residual colon devoid of epithelial cells accounted for at least 99% of the total PGs produced by intact distal colon. The profile of AA metabolites formed by submucosal structures also differed markedly from that of the epithelium. The dominant submucosal product was PGE2. PGE2 and its degradation product 13,14-dihydro-15-keto-PGE2 accounted for 63% of the PG products formed by submucosal structures (PGE2 much greater than PGD2 greater than 13,14-dihydro-15-keto-PGE2 greater than PGF2 alpha = TXB2 = 6-keto-PGF1 alpha greater than 13,14-dihydro-15-keto-PGF2 alpha). By contrast, epithelial cells, and particularly surface epithelium, contributed disproportionately to the PG degradative capacity of colon, as assessed from the metabolism of either PGE2 or PGF2 alpha. When expressed as a percentage, epithelial cells accounted for 71% of total colonic PGE2 degradative capacity but only 23% of total colonic protein. Approximately 15% of [3H] PGE2 added to the serosal side of everted colonic loops crossed to the mucosal side intact. Thus, at least a portion of the PGE2 formed in the submucosa reaches, and thereby can potentially influence functions of the epithelium.

Animals↗
PubMed1983

Effects of fluid intake on basal and vasopressin-responsive urinary prostaglandin E.

The effects of fluid intake on basal and vasopressin-responsive urinary PGE excretion (UPGEV) were examined in conscious rats under conditions of 1) ad libitum water intake, 2) water deprivation, and 3) water diuresis induced by ad libitum intake of 5% dextrose in water. UPGEV fell progressively during 40 h of water deprivation. Water diuresis after water deprivation increased UPGEV transiently (8 h). Vasopressin (Pitressin tannate in oil, 5 U/kg subcutaneously) increased UPGEV and decreased urine volume (V) in rats on ad libitum water intake but did not alter UPGEV during water deprivation. Indomethacin suppressed UPGEV (70-90%), increased basal urine osmolality (Uosmol), and potentiated the antidiuretic response to Pitressin in rats on ad libitum water intake. Indomethacin accelerated by 8 h the onset of maximal antidiuresis in water-deprived rats but did not significantly alter water balance. During water diuresis, UPGEV declined in the first 8 h after Pitressin. Thereafter, UPGEV increased markedly, concurrent with early vasopressin escape. Indomethacin or meclofenamate inhibited the rise in UPGEV, the decline in Uosmol, and the increase in V of the escape phase. Indomethacin or meclofenamate also impaired the excretion of an acute water load (5% body wt) given during escape. The spontaneous decline in UPGEV during hydropenia may serve to maximize physiologic antidiuresis. Conversely, the marked increase in UPGEV induced by administration of vasopressin during water diuresis may serve to suppress the antidiuretic response and thus play a role in the mediation of escape from physiologically inappropriate antidiuresis.

Animals↗
PubMed1983

Role of local prostaglandin synthesis in the modulation of proliferative activity of rat colonic epithelium.

The role of local prostaglandin (PG) synthesis in the modulation of the proliferative activity of colonic epithelium was examined in rat colon. Experimental rats were given either indomethacin (5 mg/kg s.c. every 8 h for three doses) or aspirin (0.5 g/100 g diet for 3 d). In rats treated with indomethacin or aspirin, the incorporation of [3H]thymidine (dThd) into DNA in vivo was increased approximately twofold over control in mucosal scrapings from distal colon, and approximately threefold over control in the proliferating pool of epithelial cells isolated from distal colon. [3H]dThd incorporation into DNA was also examined ex vivo immediately after distal colonic resection. It was approximately twofold higher in mucosa of colonic segments (1-h incubation) from rats treated with indomethacin or aspirin in vivo, compared with corresponding values of segments from control rats. Immunoreactive (i) prostaglandin E (PGE), the dominant PG product of colon segment incubates by high-performance liquid chromatography analysis of [14C]arachidonate metabolites, was markedly (95%) reduced in the media of 1-h colon incubates from indomethacin- or aspirin-treated rats, compared with control rats. Moreover, the cyclic (c)AMP content of mucosa of segments from indomethacin- or aspirin-treated rats was significantly lower than that of control rats. Prolonged incubation (4-24 h) of colonic segments from indomethacin-treated rats, in the absence of indomethacin in vitro, led to an eventual return of [3H]dThd incorporation into DNA, iPGE, and mucosal cAMP to control values. Conversely, inclusion of indomethacin (0.25 mM) in the incubations (6 h) of colonic segments from indomethacin-treated rats resulted in persistent suppression of iPGE and mucosal cAMP, as well as persistent enhancement of [3H]dThd incorporation into mucosal DNA. However, incubation of colonic segments from control rats (no in vivo drug exposure) with indomethacin or aspirin in vitro for periods up to 24 h failed to alter DNA synthesis, despite marked reduction in media iPGE and lower mucosal cAMP. The latter observations suggested that additional in vivo factors initiated the enhancement of DNA synthesis in indomethacin- or aspirin-treated rats. Exogenous PGE2, D2, I2, or F2 alpha, each of which increased the endogenous mucosal cAMP content of incubated colonic segments from control, indomethacin- or aspirin-treated rats, all suppressed [3H]dThd incorporation into mucosal DNA in vitro. Dibutyryl cAMP, but not dibutyryl cGMP, had an analogous suppressive effect on in vitro [3H]dThd incorporation into DNA. Thus, the present observations are consistent with an inhibitory action of endogenous colonic PG synthesis on the proliferative activity of colonic epithelium. This action may be mediated through cAMP.

Animals↗
PubMed1982

Evidence for independent actions of vasopressin on renal inner medullary cyclic AMP and prostaglandin E production: relationship of the prostaglandin E response to hormone pressor activity.

Arginine vasopressin (AVP) has been shown to stimulate prostaglandin (PG) production in renal medulla, while PGs have been implicated in the suppression of the antidiuretic activity of AVP. These findings have suggested a local negative feedback system involving PGs in the modulation of the antidiuretic activity of AVP. However, coupling of the antidiuretic activity of AVP to its action to increase medullary PG production is not established. In the present study of rat inner medullary slices, we concurrently examined in the same incubate the relationship between the actions of AVP to increase media immunoreactive PGE (iPGE) and tissue cAMP, the presumed first biochemical step in expression of the antidiuretic activity of the hormone. The synthetic AVP analogue [1, d(CH2)5Tyr(Me)AVP], which selectively blocks the pressor but not the antidiuretic activity of AVP, abolished the action of AVP to increase media iPGE in inner medullary incubates but did not alter AVP induced increases in tissue cAMP in slices from the same incubates. By contrast, the analogue [d(CH2)5Tyr(Et)VAVP], which blocks both the pressor and antidiuretic activity of AVP, inhibited both the cAMP and iPGE responses to AVP. The analogue 1-deamino-8-D-AVP (dD'AVP), which has potent antidiuretic activity but little if any pressor activity, markedly stimulated inner medullary cAMP accumulation without altering media iPGE. These results indicate that the acute actions of AVP to increase inner medullary cAMP and iPGE are separable and independent. The latter effect of AVP appears to be linked to the pressor rather than the antidiuretic activity of the hormone.U

Animals↗
PubMed1982

Effects of hyperosmolality on calcium mobilization in renal inner medulla: relationship to alterations in prostaglandin E synthesis.

Hypertonic NaCl and mannitol stimulate the release of [14C]arachidonate and iPGE from rat inner medullary slices, whereas hypertonic urea suppresses these same parameters. Since numerous observations indicate that arachidonate release and PG synthesis in this tissue are Ca2+-dependent processes, we examined the possibility that the divergent actions of these solutes were related to differential effects on Ca2+ availability in inner medullary slices. Inner medullary slices prelabeled with [45Ca2+] were initially perfused with 305 mOsm Krebs' buffer followed by perfusion with buffer whose osmolality was raised by the addition of 1000 mOsm NaCl, mannitol, or urea. The [45Ca2+] efflux rate coefficient, an index of Ca2+ mobilization, was determined from the release of [45Ca2+]. In Ca2+-replete buffer (1.5 mM Ca2+), addition of each solute markedly enhanced the efflux rate coefficient compared to that observed in the same slices during perfusion with 305 mOsm buffer. Addition of hypertonic NaCl, mannitol, or urea also clearly stimulated the efflux rate coefficient in slices initially perfused with Ca2+-free 305 mOsm buffer containing the Ca2+ chelator EGTA and/or La3+, which displaces superficially bound Ca2+. Moreover, each solute enhanced the efflux rate coefficient in mitochondrial fractions isolated from inner medullary slices that had been prelabeled with [45Ca2+] and initially perfused with 305 mOsm buffer. The latter observations support the ability of hypertonic NaCl, mannitol, and urea to mobilize Ca2+ bound to intracellular sites. Thus it is unlikely that the differential actions of these solutes on arachidonate release and iPGE synthesis in inner medullary slices are simple functions of their effects on Ca2+ availability. This is consistent with earlier findings which demonstrated that urea, but not NaCl or mannitol, directly inhibits Ca2+-dependent acyl hydrolase activity in plasma membrane and microsomal preparations of inner medullary slices. Accordingly, inhibitory effects of urea on PG synthesis may be related to its actions on this enzymatic step, rather than an effect on Ca2+ availability in inner medullary slices.

Animals↗
PubMed1982

Calcium-dependent stimulation of renal medullary prostaglandin synthesis by furosemide.

The present study examined the actions of furosemide and other "loop" diuretics on immunoreactive prostaglandin E (iPGE) and [14C]arachidonate (AA) release in vitro in incubates of slices from rat and dog outer or inner medulla. The loop diuretics furosemide, ethacrynic acid, bumetanide and 3-benzylamino-4-phenylthio-5-sulfamoylbenzoic acid all significantly increased [14C]AA and iPGE release (1.5- to 4-fold) into the media of rat outer and inner medulla and dog outer medullary slice incubates. By contrast, equimolar concentrations of chlorothiazide and hydrochlorothiazide were without effects on these parameters. Stimulation of [14C]AA or iPGE by furosemide was abolished by exclusion of Ca++ from the incubation media or by addition of verapamil to complete media, but was not altered by exclusion of Na+. Ca++-free media or verapamil also abolished the increases in [14C]AA and iPGE induced by ionophore A23187. By contrast, these incubation conditions did not influence the iPGE responses to hypertonic mannitol or exogenous AA. The presence of Ca++-responsive acyl hydrolase activity was demonstrated in the microsomal fraction from rat outer medulla. However, this activity was not altered by addition of furosemide to the subcellular fraction in the presence or absence of Ca++. Thus, furosemide and other loop diuretics stimulate renal medullary iPGE synthesis in vitro, and may do so through Ca++-mediated or dependent enhancement of the release of AA.

Amidohydrolases↗
PubMed1981

Cyclic nucleotide metabolism in rat colonic epithelial cells with different proliferative activities.

Cyclic nucleotide metabolism was examined in rat distal colonic epithelial cells with different proliferative activities. Lower crypt cells had DNA synthetic rates 7-10-fold higher than surface cells. Without a phosphodiesterase inhibitor proliferative cells had reduced basal cyclic AMP-, cyclic GMP-, and cyclic AMP-dependent protein kinase activity ratios, as well as blunted cyclic AMP responses to prostaglandin E2 and vasoactive intestinal peptide compared to superficial cells. In the presence of 3-isobutyl-1-methylxanthine, basal cyclic AMP and responses to prostaglandin E2 and vasoactive intestinal peptide of proliferative cells exceeded values in superficial cells. This correlated with higher membrane adenylate cyclase activity in the proliferative cells. By contrast, particulate and soluble guanylate cyclase activities of superficial cells were higher than in proliferative cells. The apparent high Km soluble and particulate cyclic AMP and cyclic GMP phosphodiesterase activities of proliferative cells were 4-7-fold higher than those in superficial cells. Moreover, the apparent low Km soluble activity was absent in superficial cells. Thus, an altered rate of nucleotide degradation may mediate reduced cyclic AMP and cyclic GMP in proliferative versus superficial cells. Dibutyryl cyclic AMP, prostaglandin E2 or vasoactive intestinal peptide inhibited [3H]thymidine incorporation into DNA of colonic segments. Thus, reduced cyclic AMP in lower crypt cells may be a determinant of their greater proliferative activity.

1-Methyl-3-isobutylxanthine↗
PubMed1981

Stimulation of rat colonic mucosal prostaglandin synthesis by calcium and carbamylcholine: relationship to alterations in cyclic nucleotide metabolism.

The present study examined the relationships between prostaglandin (PG) synthesis and cyclic nucleotide metabolism in rat colonic mucosal slices. Ca2+, Ca2+ plus A23187 and carbamylcholine all increased [14C]-arachidonate release from prelabeled slices and stimulated production of PGE. Actions of A23187 and carbamylcholine required Ca2+ and were suppressed by tetracaine or mepacrine, whose known actions include inhibition of acyl hydrolase activity. Exogenous arachidonate or linoleate stimulated PGE synthesis in the absence of Ca2+ or in the presence of the inhibitors, suggesting a role for Ca2+ dependent acyl hydrolase activity in the mediation of the actions of Ca2+, A23187 and carbamylcholine on PGE synthesis. Accumulation of both cAMP and cGMP in colonic mucosal slices was enhanced by carbamylcholine, Ca2+, Ca2+ plus A23187, arachidonate or linoleate. Stimulatory actions of each of these agents on PGE production and cyclic nucleotide accumulation were inhibited by O2 exclusion or indomethacin (100 micrograms/ml). The results support a role for local PG production in the mediation of carbamylcholine and Ca2+ actions on cyclic nucleotides. Endogenous ionic, neurohumoral and dietary factors may modulate colonic mucosal PG synthesis and cyclic nucleotide content, and thereby influence the physiologic expression of the actions of these putative local cellular regulators.

Animals↗
PubMed1981

Effects of vasopressin and urea on Ca2+-calmodulin-dependent renal prostaglandin E.

Vasopressin (AVP stimulated immunoreactive E (iPGE) synthesis and the release of [3H]arachidonate (AA) from prelabeled slices of rat inner medulla (IM) in the presence but not in the absence of Ca2+ (plus 2 mM EGTA). Urea (700-1,200 mosM) inhibited these actions. By contrast Ca2+ deprivation or urea did not suppress AVP-induced increases in cAMP or stimulation of iPGE by exogenous AA. At 10-50 microM, trifluoperazine (TFP) suppressed AVP-induced increases in [3H]AA release and iPGE accumulation, but not increases in cAMP. Basal acyl hydrolase activity (AH) of 2,000 g particulate fractions of IM was suppressed by EGTA. Ca2+, but not Mg2+ or AVP, restored AH to levels observed in the absence of EGTA. Ca2+-induced increases in particulate AH were inhibited by urea (700-1,200 mosM) or TFP (10-50 microM), but not 1,000 mosM NaCl. Partial depletion of particulate calmodulin-like activity suppressed Ca2+-induced increases in AH. The latter was restored with 1 microM purified exogenous calmodulin but not troponin C. The results demonstrate that AVP stimulation of AA and PGE release in IM are Ca2+-dependent processes suppressed by urea. They also suggest a role for Ca2+-calmodulin-dependent AH in the control of PG synthesis in IM.

Arachidonic Acid↗
PubMed1980

Calcium and O2-dependent control of inner medullary cGMP: possible role for Ca2+-dependent arachiodonate release and prostaglandin synthesis in expression of the action of osmolality on renal inner medullary guanosine 3'5' monophosphate.

The present study examined the effects of osmolality on basal cGMP metabolism and cGMP responses to carbamylcholine in rat inner medulla. The basal cGMP content of inner medullary slices and cGMP responses to carbamylcholine fell as media osmolality was increased from 305 to 1650 mosmole/liter by the addition of urea plus NaCl to standard Krebs bicarbonate buffer. Exclusion of extracellular Ca2+ or addition of tetracaine abolished the effects of both a reduction in osmolality and carbamylcholine to increase cGMP. Readdition of Ca2+ to Ca2+-deprived slices restored the actions of media osmolality and carbamylcholine on cGMP. Ionophore A23187 enhanced the effects of Ca2+ to increase slice cGMP content. Analogous to the effects of Ca2+ alone, increases in cGMP accumulation in response to Ca2+ plus A23187 were significantly suppressed at high osmolality. In slices prelabeled with [14C]-arachidonate, the stimulatory effects of Ca2+ plus A23187 on cGMP were correlated with enhanced release of [14C]-arachidonate into the media and with increased accumulation of prostaglandin E in the media, all of which were depressed in slices incubated at 1650 compared to 750 mosmole/liter. Exogenous arachidonate increased cGMP 50% to twofold in the absence of Ca2+. However, the effects of exogenous arachidonate on cGMP were clearly less than those of either Ca2+ or carbamylcholine. Addition of indomethacin or exclusion of O2 abolished effects of exogenous arachidonate, Ca2+ plus A23187, reduced osmolality, and carbamylcholine to increase cGMP. In the presence of indomethacin or in the absence of O2, Ca2+, and arachidonate-induced prostaglandin E accumulation was also not detectable. By contrast, addition of indomethacin or exclusion of O2 had no effect on Ca2+-induced [14C]-arachidonate release. Changes in cGMP accumulation in inner medulla in response to changes in media osmolality, Ca2+ plus A23187, and exogenous arachidonate were accompanied by directionally similar alterations in cAMP. Moreover, increases in cAMP induced by Ca2+ or arachidonate were inhibited by indomethacin and O2 deprivation. These data suggest that Ca2+-dependent fatty acid release and oxygenation by fatty acid release and oxygenation by cyclooxygenase may participate in the control of the metabolism of both cGMP and cAMP in inner medulla. However, PGE2 and PGI2 increased cAMP but not cGMP, whereas carbamylcholine increased cGMP but not cAMP. Thus, to the extent that arachidonate oxygenation products mediate changes in cyclic nucleotide metabolism in inner medulla, it is likely that more than one product is involved or that changes in cGMP and cAMP occur in separate compartments of inner medulla.

Animals↗
PubMed1980

Calcium-dependent action of osmolality on adenosine 3',5'-monophosphate accumulation in rat renal inner medulla: evidence for a relationship to calcium-responsive arachidonate release and prostaglandin synthesis.

When urea and NaCl are employed as the major solutes of high osmolality buffers, the cyclic AMP (cAMP) content of oxygenated slices of rat renal inner medulla increases three- to fivefold as osmolality is decreased from 1,650 to 305 mosM. Incubation of slices in Ca2+-free media containing 2 mM EGTA largely abolished this action of osmolality on cAMP, whereas exclusion of Mg2+ or 5+ from the incubation media was without effect. Addition of Ca2+ to Ca2+-deprived inner medulla incubated at 750 mosM (175 mM Na+, 380 mM urea) significantly increased tissue cAMP and media prostaglandin (PG)E accumulation. Ca2+ also stimulated the release of 14C-fatty acid from Ca2+-deprived slices prelabeled with [14C]arachidonate, but not from those labeled with [14C]palmitate. The divalent cation ionophore A23187 enhanced the actions of Ca2+ to increase tissue cAMP, media PGE accumulation, and the release of [14C]-arachidonate from prelabeled inner medulla. By contrast, when slices were incubated at 1,650 mosM (365 mM Na+, 900 mM urea) in the presence or absence of A23187, all of these actions of Ca2+ were markedly suppressed or abolished. Addition of exogenous arachidonate increased tissue cAMP and media PGE at both 750 and 1,650 mosM, whereas palmitate and stearate had no effect on cAMP at either osmolality. The actions of Ca2+ and arachidonate to increase cAMP and PGE accumulation were abolished by the cyclo-oxygenase inhibitors, indomethacin and meclofenamate. They were also abolished by exclusion of molecular O2, which serves as cosubstrate with arachidonate in prostaglandin synthesis. At maximally effective concentrations, exogenous PGE2 and arachidonate produced similar increases in inner medullary cAMP. The maximal effects of the two agents on cAMP were not additive, but were expressed in the absence of Ca2+ at both 750 and 1,650 mosM. However, in marked contrast to the O2-dependent action of arachidonate, PGE2 increased cAMP in the presence or absence of O2. Comparison of the separate actions of urea and NaCl indicated that suppression of Ca2+-responsive [14C]arachidonate release, PGE, and cAMP accumulation at 1,650 mosM reflected primarily an effect of urea, whereas hypertonic NaCl, mannitol, and sucrose alone stimulated inner medullary cAMP and PGE accumulation by a pathway which did not require extracellular Ca2+. Analogous to the actions of hypertonic urea, tetracaine and mepacrine inhibited the actions of Ca2+ plus A23187 to stimulate [14C]-arachidonate release, PGE, and cAMP accumulation. Inhibition of PGE and cAMP accumulation by tetracaine and mepacrine was also overcome by arachidonate. The results suggest that high osmolaity media with urea as a major solute reduces inner medullary cAMP content, at least in part, through effects on Ca2+-dependent prostaglandin synthesis. Inhibition of PGE synthesis, in turn, may be the result of osmotic suppression of Ca2+-dependent release of arachidonate, the availability of which is often rate limiting to prostaglandin generation.

Animals↗