Replete Medium Research Articles

The effect of folate deficiency on the hprt mutational spectrum in Chinese hamster ovary cells treated with monofunctional alkylating agents

Folic acid deficiency acts synergistically with alkylating agents to increase DNA strand breaks and mutant frequency at the hprt locus in Chinese hamster ovary (CHO) cells. To elucidate the mechanism of this synergy, molecular analyses of hprt mutants were performed. Recently, our laboratory showed that folate deficiency increased the percentage of clones with intragenic deletions after exposure to ethyl methanesulfonate (EMS) but not N-nitroso- N-ethylurea (ENU) compared to clones recovered from folate replete medium. This report describes molecular analyses of the 37 hprt mutant clones obtained that did not contain deletions. Folate deficient cells treated with EMS had a high frequency of G>A transitions at non-CpG sites on the non-transcribed strand, particularly when these bases were flanked on both sides by G:C base pairs. Thirty-three percent of these mutations were in the run of six G's in exon 3. EMS-treated folate replete cells had a slightly (but not significantly) lower percentage of G>A transitions, and the same sequence specificity. Treatment of folate deficient CHO cells with ENU resulted in predominantly T>A transversions and C>T transitions relative to the non-transcribed strand. These findings suggest a model to explain the synergy between folate deficiency and alkylating agents: (1) folate deficiency causes extensive uracil incorporation into DNA; (2) greatly increased utilization of base excision repair to remove uracil and to correct alkylator damage leads to error-prone DNA repair. In the case of EMS, this results in more intragenic deletions and G:C to A:T mutations due to impaired ligation of single-strand breaks generated during base excision repair and a decreased capacity to remove O 6-ethylguanine. In the case of ENU additional T>A transversions and C>T transitions are seen, perhaps due to mis-pairing of O 2-ethylpyrimidines. Correction of folate deficiency may reduce the frequency of these types of genetic damage during alkylator therapy.

Role of non-NMDA receptors in osmotic and glutamate stimulation of vasopressin release: effect of rapid receptor desensitization.

Previous studies demonstrated that the increase in vasopressin (VP) release and induction of VPmRNA content by osmotic stimulation was blocked by kynurenic acid, a non-specific antagonist of excitatory amino acid (EAA) receptors. In order to identify the type of EAA receptor involved, perifused explants of the hypothalamo-neurohypophyseal system (HNS) were exposed to a ramp increase in osmolality (40 mOsm over 6 h achieved by increasing NaCl) in the presence and absence of 10 microM 6,7-dinitroquinoxaline-2,3-dione (DNQX), an antagonist of non-n-methyl-d-aspartate (NMDA) excitatory amino acid receptors. Vasopressin release and VP mRNA content were significantly increased by exposure to the osmotic stimulus. 6,7-dinitroquinoxaline-2,3-dione inhibited osmotically stimulated VP release (F=16.65, P=0.0008) without significantly reducing basal release. It also prevented the osmotically stimulated increase in VP mRNA content (P <0.05). Although these results implicated glutamate, the primary endogenous ligand for EAA receptors, in the regulation of VP, exogenous glutamate was ineffective in stimulating VP release from HNS explants in either low-Mg2+ or Mg2+-replete medium. However, blockade of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor desensitization with cyclothiazide (100 microM) caused a marked increase in VP release in response to 100 microM glutamate, and blockade of kainate receptor desensitization with concanavalin A resulted in a small, but significant increase in VP release in response to 1 mM glutamate. These results support a role for non-NMDA receptor activation in osmotic regulation of VP release.

Influence of iron on growth and extracellular products of Acinetobacter baumannii.

Iron is an important nutrient required by bacteria for optimal growth. Acquisition of iron from the host where iron is restricted is an important mediator of bacterial pathogenesis. In iron deplete chemically defined medium (CDM-Fe) growth of Acinetobacter baumannii was restricted as compared to iron replete medium (CDM + Fe). Bacteria developed four high molecular weight outer membrane proteins (OMPs) of 88, 84, 80 and 77 kDa in CDM-Fe medium which were absent in CDM + Fe medium, and are known iron regulated outer membrane proteins (IROMPs). A. baumannii secreted siderophores extracellularly into the medium which act as iron chelators which had been demonstrated in the supernatants of CDM-Fe media. The siderophore was of catechol type. This shows that A. baumannii under iron restricted conditions express IROMPs along with production of catechol type siderophore in order to acquire iron from the external milieu.

Intracrine role of progesterone in fibronectin production and deposition by chicken ovarian granulosa cells in vitro: effect of extracellular calcium.

The role of extracellular Ca2+ in progesterone-induced fibronectin production and deposition by hen granulosa cells was studied. Granulosa cells isolated from preovulatory follicles of the chicken ovary were incubated in Ca(2+)-deficient or Ca(2+)-containing medium 199, and the amount of total fibronectin produced (fibronectin deposited in the matrix, secreted into the medium, or associated with cells) was measured by ELISA. The quantity of fibronectin deposited as well as the total amount of fibronectin produced in the presence or absence of exogenous progesterone was suppressed in Ca(2+)-deficient medium. It required greater concentrations of exogenous progesterone to significantly increase fibronectin production in Ca(2+)-deficient medium than in Ca(2+)-replete medium. Cyanoketone, an inhibitor of progesterone synthesis, suppressed total fibronectin produced by unstimulated cells both in the absence and presence of Ca2+. However, the inhibitory effect of cyanoketone was significantly less in Ca(2+)-replete medium than in Ca(2+)-deficient medium. Exogenous progesterone reversed completely the inhibitory effects of cyanoketone. In the presence of cyanoketone, progesterone caused a greater (3-fold) increase in fibronectin production in Ca(2+)-replete medium than in the absence of Ca2+. Thapsigargin, an agent that mobilizes Ca2+ from internal stores, suppressed basal and progesterone-induced fibronectin production in the absence and presence of cyanoketone. However, the inhibitory effect of thapsigargin was significantly reduced in Ca(2+)-replete medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of intracellular pH during cytoprotection of proximal tubule cells by glycine or acidosis.

Lowering extracellular pH to less than 7.0 strongly protects isolated proximal tubules against ATP depletion and Ca(2+)-induced injury, but there is little information about alterations of intracellular pH (pHi) in renal tubules during either injury or its modification by decreasing medium pHi or other potent protective factors such as glycine. pHi was assessed with 2',7'-bis-(2-carboxyethyl)-5-carboxyfluorescein during proximal tubule injury produced by simple ATP depletion with the electron transport inhibitor antimycin or by large increases of cytosolic free Ca2+ induced by treatment with the calcium ionophore ionomycin, alone and in combination with antimycin. Freshly isolated rabbit proximal tubules studied under superfusion conditions in the presence of probenecid were suitable for monitoring pHi during relatively prolonged and severe injury states. Probenecid, used to promote the retention of intracellular fluorophores, only minimally modified the injury response by transiently delaying lactate dehydrogenase release during antimycin treatment. The tubules did not exhibit spontaneous decreases of pHi during simple ATP depletion, but pHi fully equilibrated with cytoprotective decreases of medium pH. Irrespective of the presence of antimycin, ionomycin induced intracellular alkalinization in Ca(2+)-replete medium, which may have further enhanced the severity of injury. When medium Ca2+ was buffered to 100 nM, ionomycin induced intracellular acidification, which likely resulted from a combination of Ca2+/H+ exchange activity of the ionophore and H+ uptake during Ca(2+)-ATPase-mediated extrusion of Ca2+ released by ionomycin from intracellular pools. Alterations of pHi did not contribute to glycine cytoprotection because glycine did not affect the behavior of pHi during treatment with antimycin, ionomycin, or both agents in combination.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-adrenergic stimulation and cAMP mobilize Ca2+ from an IP3-insensitive pool in rat submandibular granular ducts.

The beta-adrenergic agonist isoproterenol induced an increase in intracellular calcium concentration ([Ca2+]i) in rat submandibular granular ducts that was blocked by beta-adrenergic but not by alpha-adrenergic or muscarinic antagonists. This effect was only partially inhibited by the selective beta 1- and beta 2-adrenergic antagonists atenolol and ICI-118,551, but was completely blocked by the combination of the two, suggesting the involvement of multiple (or atypical) beta-adrenergic receptor subtypes. The response to isoproterenol was mimicked by forskolin, 3-isobutyl-1-methylxanthine, and dibutyryl adenosine 3',5'-cyclic monophosphate, but it was blocked by protein kinase inhibitors. The response of [Ca2+]i to isoproterenol was sustained in Ca(2+)-replete replete medium but transient in Ca(2+)-free medium, indicating the involvement of both Ca2+ entry and release from intracellular stores. However, isoproterenol stimulation produced no increase in ductal inositol phosphate levels. In addition, isoproterenol was still able to increase [Ca2+]i after the carbachol-induced depletion of inositol 1,4,5-trisphosphate (IP3)-sensitive calcium stores. We conclude that isoproterenol, acting through cAMP, releases Ca2+ from an IP3-insensitive intracellular store in salivary granular ducts.

Intracellular calcium and the signaling mechanism of luteinizing hormone—releasing hormone in rat granulosa cells

The purpose of these studies was to determine the source(s) of the increase in intracellular free calcium in response to luteinizing hormone-releasing hormone in ovarian granulosa cells. Rat granulosa cells were cultured and loaded with fura-2-acetoxy-methyl ester, a fluorescent calcium indicator dye, and intracellular free calcium measured by microspectrofluorometry. The source of the luteinizing hormone-releasing hormone induced increase in intracellular Ca++ was investigated with various calcium channel blockers (verapamil, diltiazem, and nifedipine), high K+ buffer, and perifusion with media lacking Ca++. All three voltage-sensitive calcium channel blockers (10(-5) mol/L) tested were ineffective in blocking the luteinizing hormone-releasing hormone induced intracellular Ca++ increase. Treatment with high K+ buffer also had no effect. Perifusion with media lacking calcium resulted in a gradual loss of the luteinizing hormone-releasing hormone response, an effect that was accelerated by repeated stimulation with hormone. Transient replacement of extracellular Ca++ failed to restore the response but continued perifusion with Ca(++)-replete media allowed a luteinizing hormone-releasing hormone response 10 minutes later. The luteinizing hormone-releasing hormone induced intracellular Ca++ increase does not appear to result from the opening of voltage-sensitive or K(+)-dependent Ca++ channels. To the contrary, this response likely results from the release of Ca++, primarily from intracellular stores.

Bumetanide-sensitive NaCl uptake in rabbit tracheal epithelial cells is stimulated by neurohormones and hypertonicity.

Loop diuretic-sensitive NaCl(K) cotransport plays a fundamental role in absorption and secretion of electrolytes in epithelial tissues. Cotransport activity was measured as uptake of 22Na, 36Cl, or 86Rb at 27 degrees C in isolated rabbit tracheal epithelial cells. Uptake of radiotracer was linear from 1 to 2 min after initiation of radiotracer transport. Bumetanide at 10 microM final concentration did not affect tracer uptake. The endogenous catecholamine l-epinephrine and alpha 2-adrenergic agent clonidine increased sodium and chloride uptake at least 5.5-fold. Bumetanide blocked sodium uptake by 85% and chloride uptake by 72%. 86Rb uptake was not affected by l-epinephrine, clonidine, or bumetanide. The alpha 2-adrenergic antagonist yohimbine blocked the effects of l-epinephrine and clonidine on 22Na and 36Cl uptake. In Ca(2+)-depleted transport medium, baseline levels of sodium and chloride uptake increased 3.8- and 2.4-fold, respectively, in a bumetanide-independent manner. Nevertheless, l-epinephrine and clonidine induced a net stimulation of sodium and chloride uptake similar to that found in Ca(2+)-replete medium. This response was reduced by bumetanide and yohimbine. The Ca(2+)-elevating agent ionomycin increased bumetanide-sensitive sodium and chloride uptake 7.2- and 6.2-fold, respectively. Replacement of chloride with gluconate or sodium with N-methyl-D-glucamine in the extracellular medium inhibited l-epinephrine and clonidine-stimulated bumetanide-sensitive sodium and chloride uptake, respectively. Osmotic shrinkage in hyperosmotic (500 mM NaCl with all other electrolytes at normal concentration) transport medium markedly increased bumetanide-inhibitable sodium and chloride uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

The Na+/Ca2+ exchanger regulates cytolysin/perforin-induced increases in intracellular Ca2+ and susceptibility to cytolysis.

The Ca2+ dependency of NK cell-mediated and cytolysin-mediated cytolysis may be related to increases in target cell intracellular Ca2+. In a previous study we hypothesized that the Na+/Ca2+ exchanger can act as a counter-lytic mechanism by regulating the damaging increases in intracellular free calcium ([Ca2+]i) produced by cytolysin. We found that conditions said to inhibit Ca2+ extrusion by Na+/Ca2+ exchange, namely low extracellular Na+ or the presence of certain amiloride analogs which block Na+/Ca2+ exchange, enhanced the cytolysin-mediated cytolysis of YAC-1 lymphoma cells. In the present work we have confirmed the above hypothesis by measuring the [Ca2+]i of fura-2- or aequorin-labeled YAC-1 cells treated with cytolysin and low Na+ medium or amiloride analogs. YAC-1 cells appear to have a Na+/Ca2+ exchange system: low Na+ medium caused gradual increases in [Ca2+]i, and this effect was reversed in Na(+)-replete medium. Cytolysin purified from NK cell granules caused rapid dose-dependent increases in [Ca2+]i, and low Na+ medium enhanced these cytolysin-mediated increases. The Na+/Ca2+ exchange system appeared to be more active in cytolysin-challenged cells: amiloride analogs, which inhibit Na+/Ca2+ exchange in other systems, acted synergistically with cytolysin to cause large increases in [Ca2+]i, but had little effect, if any, on their own. 5-(N-4-Chlorobenzyl)-2',4'-dimethylbenzamil, the amiloride analog which has the greatest specificity for the Na+/Ca2+ exchanger and which previously was found to be the most potent enhancer of cytolysin-mediated cytolysis, was the most potent enhancer of cytolysin-mediated increases in [Ca2+]i. The above results suggest that Na+/Ca2+ exchange may be one of the target cell mechanisms of resistance to cytolysin and NK cell-mediated cytolysis.

Mutants of human colon adenocarcinoma, selected for thymidylate synthase deficiency.

GC3/c1 human colon adenocarcinoma cells were treated with the mutagen ethyl methanesulfonate, and three clones deficient in thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1. 45) activity were selected and characterized. Growth in medium deficient in thymidine caused cell death in two clones (TS- c1 and TS- c3), whereas one clone (TS- c2) showed limited growth. Growth correlated with thymidine synthase activity and 5-fluoro-2'-deoxyuridine 5'-monophosphate-binding capacity and with incorporation of 2'-deoxy[6-3H]uridine into DNA. In the presence of optimal thymidine, growth rates were only 5-18% that of the parental clone (GC3/c1), which grew equally well in thymidine-deficient or -replete medium. Analysis of poly(A)+ RNA showed normal levels of a 1.6-kilobase transcript in TS- c1 and TS- c2 but decreased levels (approximately 6% control) in TS- c3. Clone TS- c3 was 32-, 750-, and greater than 100,000-fold more resistant than the parental clone to 5-fluorouracil, 5-fluoro-2'-deoxyuridine, and methotrexate, respectively. When inoculated into athymic nude mice, each TS- clone produced tumors, demonstrating continued ability to proliferate in vivo.

The role of HCO3- and Na+/H+ exchange in the response of rat parotid acinar cells to muscarinic stimulation.

The intracellular pH (pHi) of a rat parotid acinar preparation was monitored using the pH-sensitive fluorescent dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Under resting (unstimulated) conditions both Na+/H+ exchange and CO2/HCO3- buffering contribute to the regulation of pHi. Muscarinic stimulation (carbachol) of the acini produced a gradual rise in pHi (approximately 0.1 unit by 10 min) possibly due to activation of the Na+/H+ exchanger. When the exchanger was blocked by amiloride or sodium removal, carbachol induced a dramatic (atropine inhibitable) decrease in pHi (approximately 0.4 pH unit with t1/2 approximately 0.5 min at 1 mM carbachol). The rate of this acidification was reduced by removal of exogenous HCO3- and by the carbonic anhydrase inhibitor methazolamide. Also, acini stimulated with carbachol in Cl- -free solutions showed a more pronounced acidification than in the corresponding Cl- -replete media. Taken together, these data indicate that the carbachol-induced acidification of rat parotid acinar cells unmasked by inhibition of the Na+/H+ exchanger is due to a rapid loss of intracellular HCO3-. Carbachol induced acidification was inhibited by the Cl- channel blocker diphenylamine 2-carboxylate but not by 4-acetomido-4'-isothiocyanostilbene-2,2'-disulfonic acid, an inhibitor of Cl-/HCO3- exchange. In addition, this acidification could not be sustained in Ca2+-free media and was totally blocked by chelation of intracellular Ca2+. Interpreted in terms of HCO3- loss, these results closely parallel the pattern of carbachol-induced Cl- release from this same preparation and indicate that HCO3- is secreted in response to muscarinic stimulation via the same or a very similar exit pathway, presumably an apical anion channel. Under normal physiological conditions the intracellular acidification resulting from HCO3- secretion is buffered by the Na+/H+ exchanger.

Bradykinin increases cytosolic free [Ca2+] in proximal tubule cells.

The role of bradykinin (BK) as a calcium-mobilizing agonist in cells of renal proximal tubule origin was examined. Experiments were performed on confluent cultures of rabbit proximal tubule cells in primary culture and changes in cytosolic free Ca2+ concentration, [Cai2+], were monitored by use of the Ca2+-sensitive fluorescent probe fura-2. Under steady-state conditions, [Cai2+] was 210 +/- 7 nM in a Ca2+-replete medium vs. 135 +/- 5 nM in a medium devoid of Ca2+. Acute challenge with BK resulted in a transient increment in [Cai2+], which peaked at 150% the resting value within 10 s and was independent of medium [Ca2+]. The K0.5 for the process was 2.5 X 10(-10) M. The BK receptor displayed properties of the beta 2-variety. In a Ca2+-free medium 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) blocked the BK-elicited Ca2+ transient in a time- and dose-dependent manner. In contrast to TMB-8, the Ca2+-channel inhibitor, verapamil, was without effect. Prior exposure of cells to ionomycin completely obviated the BK-induced Ca2+ transient. Cells challenged with BK were nonresponsive to subsequent challenge by a second Ca2+-mobilizing agonist, angiotensin II (ANG II). In summary, these data suggest that BK is an extremely sensitive activator of the phosphoinositol transduction pathway in rabbit proximal tubule cells. Furthermore, the heterologous desensitization between BK and ANG II, in terms of elevating [Cai2+], suggests that these two agonists release Ca2+ from a common intracellular store.

Mechanism of the antihypertensive effect of K depletion in the spontaneously hypertensive rat

K depletion reverses hypertension in the SHR (systolic blood pressure: K deplete 122 +/- 5 vs. K replete 164 +/- 4 mm Hg, P less than 0.001). To determine the role of the renin angiotensin system in the protective effect of K depletion, we performed studies of vascular reactivity in intact SHR and of angiotensin II (Ang II) binding to mesenteric artery particles and vascular smooth muscle cells (VSMC) in culture from SHR. Pressor sensitivity to Ang II (+/- converting enzyme inhibition) but not norepinephrine was reduced in K depleted SHR. Thus, the decreased vascular reactivity in K depletion was specific for Ang II rather than a generalized defect. Ang II binding and receptor number (Bmax) (156 +/- 20 vs. 81 +/- 5 fmol/mg of protein, P less than 0.01) were increased in K depleted mesenteric artery particles. Since K depletion and increases in Ang II have both been associated with increased Ang II binding, Ang II binding was measured after bilateral nephrectomy. Despite reduction of plasma renin activity, total binding and Bmax were still increased in nephrectomized K depleted SHR. To determine the specific effect of K depletion independent of Ang II on Ang II binding, studies were performed in mesenteric artery VSMC from SHR grown in culture. VSMC from K replete SHR were grown to confluency in K replete medium and then were incubated in K depleted medium for 24 hours. Binding was saturable, time and temperature-dependent in K replete and K depleted cells. Total binding and Bmax (139 +/- 13 vs. 93 +/- 7 fmol/mg protein, P less than 0.01) were increased in K depleted cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cl-/HCO3- exchange modulates intracellular pH in rat type II alveolar epithelial cells.

The role of an anion exchange pathway in modulating intracellular pH (pHi) under steady-state and alkaline load conditions was investigated in confluent monolayers of rat type II alveolar epithelial cells using the pH-sensitive fluorescent probe 2'-7'-biscarboxy-ethyl-5,6-carboxylfluorescein. Under steady-state conditions in the presence of 25 mM HCO3-, 5% CO2 at pHo 7.4, pHi was 7.32 in a Na+-replete medium and 7.33 in the absence of Na+. Steady-state pHi was 7.19 in a nominally HCO3(-)-free medium at pHo 7.4, and 7.52 in a Cl(-)-free medium, with both values significantly different from that obtained in the presence of both HCO3- and Cl-. Monolayers in which pHi was rapidly elevated by removal of HCO3-/CO2 from the bathing medium demonstrated an absolute requirement for Cl- to recover toward base-line pHi. The Km of Cl- for the external site of the exchange pathway was 11 +/- 1 mM. Recovery of pHi from the alkaline load in the presence of Cl- was inhibited 60% by the stilbene derivative 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Removal of Cl- from the medium of cells bathed in HCO3-/CO2 resulted in a rapid increment in pHi which returned to base line when Cl- was reintroduced into the bathing medium. In contrast, pHi was not perturbed by removal or addition of Cl- to monolayers bathed in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered medium, indicating that HCO3- was the preferred species for transport. Recovery of pHi from an alkaline load was not affected by the presence or absence of Na+. These findings define the transport pathway as Na+-independent Cl-/HCO3- exchange. This pathway contributes importantly to determining resting pHi of pneumocytes and enables the cell to recover from an alkaline load.

Effects of bradykinin and angiotensin II on intracellular Ca2+ dynamics in endothelial cells.

The purpose of this study was to investigate the effects of angiotensin II and bradykinin on intracellular Ca2+ dynamics in cultured endothelial cells. We used the "second-generation" fluorescent Ca2+ indicator fura-2, in conjunction with dual-wavelength fluorescence spectroscopy, in cultured adherent pulmonary arterial endothelial cells. Angiotensin II (up to 2 microM) had no consistent effect on intracellular Ca2+ levels. In contrast, bradykinin (10 nM) elicited a transient increase of cytosolic free Ca2+, from the resting value of 37 +/- 5 to 647 +/- 123 nM, followed by a decline to a steady-state value of 113 +/- 14 nM, which was significantly higher than the resting Ca2+ levels. Bradykinin's Ca-stimulatory effect was dose dependent, having a half-maximally effective concentration of approximately 1 nM and a maximally effective concentration of 10 nM. A B1-receptor agonist, Des-Arg9-bradykinin, was much less effective than bradykinin as modulator of cytosolic Ca2+. Moreover, a B1-receptor antagonist, Des-Arg9, [Leu8]-bradykinin, did not significantly affect the increase of cytosolic Ca2+ elicited by bradykinin. On the other hand, the bradykinin-elicited increase of Ca2+ was almost completely inhibited by a novel B2-receptor antagonist, D-Arg-[Hyp3, Thi5,8, D-Phe7]-bradykinin. Bradykinin increased cytosolic free Ca2+ levels in cells maintained in Ca2+-deficient extracellular medium, suggesting that the peptide mobilized Ca2+ from intracellular stores. However, the absence of extra-cellular Ca2+ resulted in an 80-90% attenuation of the transient Ca2+ response, whereas the posttransient steady-state response was completely absent. These findings are consistent with the notion that the bradykinin-elicited transient Ca2+ response is dependent on both extra- and intracellular Ca2+ and that the posttransient steady-state response is entirely dependent on extracellular Ca2+. Endothelial cells were responsive to a second dose of bradykinin after a 10-min interim period of incubation in the absence of the peptide hormone. The absence of extracellular Ca2+ during the interim period, or the pretreatment of cells with ionomycin in the absence of extracellular Ca2+, prevented the response of the cells to a second dose of bradykinin. Bradykinin- or ionomycin-desensitized cells could be resensitized by a brief incubation period in Ca2+-replete medium. The results are consistent with the notions that cellular resensitization requires the replenishment of intracellular Ca2+ and that bradykinin, but not angiotensin II, modulates intracellular Ca2+ dynamics in endothelial cells by interacting with a B2-type receptor.

PHYSIOLOGICAL AND ENVIRONMENTAL CONTROL OF GERMINATION INSCRIPPSIELLA TROCHOIDEA(DINOPHYCEAE) RESTING CYSTS1

ABSTRACTThe effects of aging, temperature, and growth medium on germination in culture‐produced resting cysts of the marine dinoflagellate Scrippsiella trochoidea (Stein) Loeblich are examined. Cysts undergo a mandatory period of dormancy lasting approximately 25 days, during which germination does not occur. The duration of this period is not affected by temperature. Once the dormancy period is completed, germination is regulated by external factors. Cysts germinate optimally in nutrient replete medium at temperatures greater than approximately 14°C. At lower temperatures or in nutrient‐depleted media germination rate is dramatically slowed, although the final germination frequency appears unchanged. The large Q10of this temperature effect (ca. 11) suggests that the reduction in germination rate at lower temperatures is not merely the reflection of generally reduced metabolic rates, but rather the result of a temperature response specific to germination. At the highest temperatures tested (22‐25°C), germination rate remains maximal although vegetative growth is greatly reduced. A shift in temperature or nutrient conditions, per se, is not necessary for germination. The relatively short dormancy period combined with the absence of a requirement for a dramatic shift in environmental conditions could facilitate rapid cycling between resting and vegetative stages in natural S. trochoidea populations. At the same time, the dramatic reduction in germination rate at low temperatures would permit cysts of this species to serve as overwintering cells as well.

Interactions of sodium transport, cell volume, and calcium in frog urinary bladder.

The volume of individual cells in intact frog urinary bladders was determined by quantitative microscopy and changes in volume were used to monitor the movement of solute across the basolateral membrane. When exposed to a serosal hyposmotic solution, the cells swell as expected for an osmometer, but then regulate their volume back to near control in a process that involves the loss of KCl. We show here that volume regulation is abolished by Ba++, which suggests that KCl movements are mediated by conductive channels for both ions. Volume regulation is also inhibited by removing Ca++ from the serosal perfusate, which suggests that the channels are activated by this cation. Previously, amiloride was observed to inhibit volume regulation: in this study, amiloride-inhibited, hyposmotically swollen cells lost volume when the Ca++ ionophore A23187 was added to Ca++-replete media. We attempted to effect volume changes under isosmotic conditions by suddenly inhibiting Na+ entry across the apical membrane with amiloride, or Na+ exit across the basolateral membrane with ouabain. Neither of these Na+ transport inhibitors produced the expected results. Amiloride, instead of causing a decrease in cell volume, had no effect, and ouabain, instead of causing cell swelling, caused cell shrinkage. However, increasing cell Ca++ with A23187, in both the absence and presence of amiloride, caused cells to lose volume, and Ca++-free Ringer's solution (serosal perfusate only) caused ouabain-blocked cells to swell. Finally, again under isosmotic conditions, removal of Na+ from the serosal perfusate caused a loss of volume from cells exposed to amiloride. These results strongly suggest that intracellular Ca++ mediates cell volume regulation by exerting a negative control on apical membrane Na+ permeability and a positive control on basolateral membrane K+ permeability. They also are compatible with the existence of a basolateral Na+/Ca++ exchanger.

PHYSIOLOGICAL AND ENVIRONMENTAL CONTROL OF GERMINATION INSCRIPPSIELLA TROCHOIDEA(DINOPHYCEAE) RESTING CYSTS1

ABSTRACTThe effects of aging, temperature, and growth medium on germination in culture‐produced resting cysts of the marine dinoflagellateScrippsiella trochoidea(Stein) Loeblich ore examined. Cysts undergo a mandatory period of dormancy lasting approximately 25 days, during which germination does not occur. The duration of this period is not affected by temperature. Once the dormancy period is completed, germination is regulated by external factors. Cysts germinate optimally in nutrient replete medium at temperatures greater than approximately 14° C. At lower temperatures or in nutrient‐depleted media germination rate is dramatically slowed, although the final germination frequency appears unchanged. The large Q10of this temperature effect (ca. 11) suggests that the reduction in germination rate at lower temperatures is not merely the reflection of generally reduced metabolic rates, but rather the result of a temperature response specific to germination. At the highest temperatures tested (22–25° C), germination rate remains maximal although vegetative growth is greatly reduced. A shift in temperature or nutrient conditions, per se, is not necessary for germination. The relatively short dormancy period combined with the absence of a requirement for a dramatic shift in environmental conditions could facilitate rapid cycling between resting and vegetative stages in naturalS. trochoideapopulations. At the same time, the dramatic reduction in germination rate at low temperatures would permit cysts of this species to serve as overwintering cells as well.

36Cl fluxes in dispersed rat submandibular acini: effects of Ca2+ omission and of the ionophore A23187.

Transmembrane fluxes of 36Cl were investigated in dispersed acini of the rat submandibular gland in Ca2+-containing and Ca2+-free media and also in the presence of the divalent cation ionophore A23187. In Ca2+-replete medium, a time-dependent uptake of tracer resulted in a steady state 36Cl content of 8.5 +/- 0.3 nmol/mg protein in 3-5 min. This uptake was reduced 32% by 1 mM furosemide and 27% by 1 microM acetylcholine. In the presence of Ca2+, the ionophore (10(-5) M) reduced tracer uptake 36% and prevented further effects of either acetylcholine or furosemide. Both acetylcholine and A23187 caused a rapid net efflux of 36Cl from tracer-preloaded acini in Ca2+-containing medium (37% and 20%, respectively). When Ca2+ was omitted from the incubation medium, basal 36Cl uptake in the absence of added test substance was the same as in Ca2+-containing medium but was not affected by acetylcholine, while it was still reduced 29% by furosemide. Addition of acetylcholine to preloaded acini in Ca2+-free medium caused only a transient and unsustained 36Cl efflux but subsequent addition of Ca2+ produced a 36% reduction in tracer content. The ionophore caused a net 36Cl efflux in Ca2+-containing medium (24% decrease in 36Cl content) but had no effect in Ca2+-free medium. Subsequent addition of Ca2+ resulted in a 27% net efflux of tracer. The calmodulin inhibitor trifluoperazine caused a 14% increase in 36Cl uptake but did not cause 36Cl efflux from preloaded cells or modify acetylcholine-induced efflux of tracer from these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium gradient-driven transport processes in ATP-depleted renal tubules.

The effects of directed Na+ gradients on proximal tubule cell transport processes were examined in suspensions of rabbit renal tubules depleted of ATP. Cells of high-Na+ content were generated by suspending the tubules in high-Na+ media, whereas low-Na+ cells were produced by incubating tubules in Na+-free media. Resuspension of the high-Na+ tubules in Na+-free media caused a fall in cell pH simultaneous with a fall in cell Na+. Resuspending the low-Na+ cells in Na+-replete media led to a rise in cell pH, in parallel with the rise in cell Na+. Removing HCO-3 and CO2 augmented and amiloride inhibited the increase in cell pH generated by the inward Na+ gradient. Low-Na+ tubules exposed to an inwardly directed Na+ gradient also concentrated the sugar analogue alpha-methylglucoside, and this uptake was blocked by phlorizin. These findings demonstrate the suitability of ATP-depleted renal tubules for the study of linked transport processes by providing evidence for the existence of the proximal luminal transport processes, Na+-H+ exchange, and Na+-sugar cotransport in this preparation.