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We have documented that both receptors of angiotensin II (ANG II) (AT1 and AT2) are involved in regulation of intracellular signals in glomerular epithelial cells (GECs). We studied the role of these receptors in regulation of intracellular ionized calcium [Ca2(+)]i in GECs. Cells were loaded with Indo-1 (Ca2(+)) and SNARF-1 (pH) fluorescent dyes and then incubated with or without ANG II for 1 hour at 37 degrees C. In some experiments AT(1) and AT(2) receptor blockers (Losartan and PD 12339, respectively) were added. In additional experiments cells were incubated with thapsigargin (Tg) and bradykinin (BK) as well as ANG II. A four-channel fluorescence videomicroscope system was used to measure real-time [Ca2(+) ]i in individual cells. Levels of inositol triphosphate (IP(3)) were measured with radioimmunoassay. An amount of 100 nmol/L of ANG II caused a maximal increase in [Ca2(+)]i in calcium-containing buffer. ANG II had no effect on intracellular pH of GECs. Increase in [Ca2(+)]i by ANG II was prevented by the concurrent use of Losartan and PD 123319. BK caused a transient increase in [Ca2(+)]i, which was significantly decreased by ANG II; concurrent addition of Losartan and PD 123319 prevented ANG II effect. ANG II prevented the accumulation of Ca2(+) in intracellular stores. ANG II caused a significant but transient increase in levels of IP(3). In summary, ANG II increases extracellular/intracellular calcium dependent bidirectional Ca2(+) transport in GECs, inhibits BK induced release of Ca2(+) from IP(3) sensitive stores, and, in addition, reduces refilling of endoplasmic reticulum [Ca2(+)] depleted by repeated BK stimulation. Both receptor subtypes appear to be important in ANG II mediated physiologic responses of GECs and may participate in modulation of glomerular function in vivo.

Thrombin, the ultimate enzyme in the blood coagulation cascade, has prominent actions on various cells, including neurons. As in platelets, thrombin increases [Ca2+]i mobilization in neurons, and also retracts neurites. Both these effects are mediated through a G protein-coupled, proteolytically activated receptor for thrombin (PAR-1). Prolonged exposure to thrombin kills neurons via apoptosis, that may also involve PAR-1 activation. Increased [Ca2+]i has been a unifying mechanism proposed for cell death in several neurodegenerative diseases. Thrombin-elevated calcium levels may activate intracellular cascades in neurons leading to cell death. Since thrombin mediates its diverse effects on cells through both heterotrimeric and monomeric G proteins, we also explored what effect altering differential G protein coupling would have on the neuronal response to thrombin. We studied calcium mobilization by thrombin in a model motor neuronal cell line, NSC19, using fluorescence image analysis. Confirming effects in other neuronal types, thrombin caused dramatic increases in [Ca2+]i levels, both transiently and after prolonged exposure, which involved activation and cleavage of the PAR-1 receptor. Using enzyme linked immunosorbent assay (ELISA) and dot-blot analysis, we found that the N-terminal fragment of PAR-1 was released into the medium after exposure to thrombin. We confirmed that PAR-1 protein and mRNA expression occurred in motor neurons. We found that cholera toxin inhibited thrombin-mediated Ca2+ influx, pertussis toxin did not significantly alter thrombin action, and lovastatin, a small 21-kDa Ras GTPase (Rho) modulator, showed a tendency to reduce the thrombin effect. These data indicate that thrombin-increased [Ca2+]i, sufficient to trigger cell death in motor neurons, might be approached in vivo by modulating thrombin signaling through PAR-1.

Intracellular calcium ([Ca2+]i) and hydrogen ion concentrations (pHi) are important regulators of cell function. Those ions also may interact and it is important, therefore, to measure their concentrations simultaneously. In the present studies we used a system developed for that purpose, a fluorescent emission ratio technique for simultaneous analysis of calcium (Indo-1) and pH (SNARF-1) in single cells at video rates, and determined if arginine vasopressin (AVP, 12.5 mumol/l) evoked [Ca2+]i and pHi signals interact in MDCK cells. We also employed a simple system for analysing the side specific (basolateral or apical) application of agonist to polarized cell layers on permeable membranes. AVP is found to evoke simultaneous changes in both pHi and [Ca2+]i. Basolateral application induced transient acidification, followed by partial recovery, and a [Ca2+]i transient with kinetic pattern similar to that of the pHi. Apical application also caused a mirror image pHi and [Ca2+]i pattern but of smaller magnitude (no peak). Selective removal of extracellular calcium ([Ca2+]e) or sodium ([Na+]e) dissociated the pHi and [Ca2+]i responses in both cases. Na+e removal abolished the pHi changes, but not the [Ca2+]i transients. [Ca2+]e removal abolished the [Ca2+]i changes and reduced, but did not abolish, the pHi responses. Thus, AVP induces pHi changes which are modified by calcium while calcium signalling is not modified by changes in pHi.

Bradykinin (BK) induces intracellular calcium ([Ca2+]i) release in Madin-Darby canine kidney (MDCK) cells. During long-term continuous BK exposure, cells become desensitized and fail to respond to a new BK stimulus. We used a protocol of repeated short-term BK addition and removal. MDCK cells were loaded with the Ca-indicator indo-1 and were exposed to BK (100 nmol/L) for 10 seconds, followed by BK removal. This cycle was repeated four to eight times while [Ca2+]i was continuously recorded. In a Ca-free bath, the cells gradually became completely desensitized to repeated BK stimuli. In the presence of 1 mmol/L or 10 mmol/L Cae, however, repeated addition of BK caused repeated [Ca2+]i transients with partial decrease of peak heights (327 and 436 nmol/L delta[Ca2+]i final) (partial desensitization). Repeated BK stimuli also led to partial desensitization (70% to 85%) to adenosine triphosphatase and carbachol (heterologous desensitization). BK also reduced peak thapsigargin response (70%), consistent with partial depletion of endoplasmic reticulum Ca pools. Our results show that MDCK cells maintain their sensitivity to BK during repeated short-term BK exposures. Available Ca3 plays a major role in modulating the degree of cellular responsiveness.

Our recent study has shown that a calcitonin (CT)-like immunoreactive substance(s) is secreted by cultured prostate cells, and secretion of this material is significantly higher in malignant than in benign prostate cells. To test the hypothesis that prostatic CT may serve as a paracrine/neuroendocrine factor, the present study investigated for the presence of CT receptors in the prostate gland. Signal transduction mechanisms activated by CT were examined, and the study also tested its effects on prostate cell proliferation, as assessed by [3H]thymidine incorporation. The results show that high affinity binding sites for [125I]salmon CT were present in plasma membrane fractions of human prostate tissue specimens and the prostate cancer LnCaP cell line. The maximal binding for CT receptors was 564 +/- 163 fmol/mg protein, and the apparent dissociation constant (Kd) was 2.89 +/- 0.58 nM. CT induced a dose-dependent increase in cAMP generation in LnCaP cells. The effect of CT on cytoplasmic Ca2+ transients of LnCaP cells was examined by videofluoromicroscopy. CT (100 nM) induced a rapid and sharp increase in cytoplasmic Ca2+ concentrations in LnCaP cells. The CT-induced increase in cytoplasmic Ca2+ transients appeared to be biphasic (spike and plateau), and this increase was 4- to 10-fold during the initial phase. The profile of this response is characteristic of the activated Ca2+/phospholipid second messenger system. CT also caused a dose-dependent increase in [3H]thymidine incorporation by LnCaP cells. These results suggest that a locally secreted CT-like peptide(s) induces mitogenic responses in prostate cancer cells. This action seems to be mediated through activation of signaling mechanisms, leading to the accumulation of two different second messengers, cAMP and calcium. Activation of dual second messenger systems by CT receptors suggests that the peptide hormone may play an important role in rapidly growing cell populations during the process of tumor formation.

The interrelationships between changes in intracellular calcium concentration ([Ca2+]i) and intracellular pH in Madin-Darby canine kidney cells and kidney glomerular epithelial cells exposed to various stimuli were analyzed simultaneously using a new design of a fluorescence video microscope. Cells were double labeled with indo 1 and SNARF 1 dyes and were excited simultaneously at 350 and 540 nm. Images at four emission wavelengths were captured simultaneously at 405, 475, 575, and 640 nm at 30 frames/s for the two ratio dyes. SNARF sensitivity to pH between 6.5 and 8.0 was unchanged by [Ca2+]i. The SNARF ratio maps were used to correct the pH-dependent changes in the calculation of local cell calcium. NH4Cl loading produced the expected alkalinization and a concurrent rise in [Ca2+]i. When the NH4Cl was removed and the cells became acidic, a second rise in [Ca2+]i was recorded. Both changes in [Ca2+]i were from intracellular stores since they persisted in the absence of extracellular calcium. The findings demonstrate the need for pH correction of indo 1 recordings.