Article date: July 1996
By: M. Reza Zamani, David R. Bristow, in Volume 118, Issue 5, pages 1119-1126
In GT1‐7 cells, histamine stimulated the initial [Ca2+]i transient in a dose‐dependent manner with a best‐fit EC50 value of 4.2 ± 4.2 μm (mean ± s.e.mean, n = 4) and a best‐fit maximal effect of 138 ± 56 nM (n = 4) increase above basal calcium levels.
Pretreatment of cells with 30 μm histamine for 30 min desensitized the population mean peak calcium signal by 53% to 75 ± 9 nM, (n = 3, P < 0.04). Analysis of the individual cells revealed that 39 ± 7% (n = 94 cells from 8 experiments) of pretreated cells exhibited desensitized histamine‐stimulated [Ca2+]i transients of ≤ 1 standard deviation below the control cells mean calcium transient level.
The desensitization induced by histamine was prevented (P < 0.01) by KN‐62 (10 μm), a putative inhibitor of the calcium/calmodulin‐dependent protein kinase II (CaMKII). KN‐62 (10 μm) alone did not induce [Ca2+]i mobilization, nor did it antagonize the histamine‐stimulated [Ca2+]i signal. In addition, KN‐62 did not appear to have its effect by hastening the rate of recovery from desensitization.
Histamine pretreatment in nominal (zero calcium + 0.2 mM EGTA) or in low (0.3 mM) extracellular calcium did not induce histamine receptor desensitization, supporting a role for extracellular calcium in the homologous H1 receptor desensitization process.
Histamine (30 μm) stimulated at least four different types of [Ca2+]i signals in GT1‐7 cells. The majority (61%) were of single spikes with the remaining cells showing some form of calcium oscillatory behaviour. The proportion of GT1‐7 cells showing histamine‐induced calcium oscillations was histamine concentration‐dependent and significantly reduced after acute desensitization. KN‐62, when present during histamine pretreatment, prevented this fall in calcium oscillation. Under the conditions of nominal or 0.3 mM extracellular calcium the proportion of cells exhibiting histamine‐stimulated calcium oscillations was not significantly different from the controls.
Bradykinin stimulated a [Ca2+]i transient in GT1‐7 cells with a population mean peak response of 147 ± 8 nM (n = 5) over basal levels. The bradykinin‐induced [Ca2+]i signal was without any calcium oscillatory activity. Histamine pretreatment caused the heterologous desensitization of the bradykinin [Ca2+]i signal (44% reduction, P < 0.007), which was unaffected by KN‐62.
The results presented here suggest that the histamine‐mediated homologous H1 receptor desensitization process involves extracellular calcium and can be blocked by KN‐62, a putative inhibitor of CaMKII. In contrast, KN‐62 does not appear to prevent the histamine‐mediated heterologous desensitization cascade. These findings suggest fundamental differences in the mechanisms underlying homologous and heterologous H1 receptor desensitization pathways in GT1‐7 neuronal cells.
In GT1‐7 cells, histamine stimulated the initial [Ca2+]i transient in a dose‐dependent manner with a best‐fit EC50 value of 4.2 ± 4.2 μm (mean ± s.e.mean, n = 4) and a best‐fit maximal effect of 138 ± 56 nM (n = 4) increase above basal calcium levels.
Pretreatment of cells with 30 μm histamine for 30 min desensitized the population mean peak calcium signal by 53% to 75 ± 9 nM, (n = 3, P < 0.04). Analysis of the individual cells revealed that 39 ± 7% (n = 94 cells from 8 experiments) of pretreated cells exhibited desensitized histamine‐stimulated [Ca2+]i transients of ≤ 1 standard deviation below the control cells mean calcium transient level.
The desensitization induced by histamine was prevented (P < 0.01) by KN‐62 (10 μm), a putative inhibitor of the calcium/calmodulin‐dependent protein kinase II (CaMKII). KN‐62 (10 μm) alone did not induce [Ca2+]i mobilization, nor did it antagonize the histamine‐stimulated [Ca2+]i signal. In addition, KN‐62 did not appear to have its effect by hastening the rate of recovery from desensitization.
Histamine pretreatment in nominal (zero calcium + 0.2 mM EGTA) or in low (0.3 mM) extracellular calcium did not induce histamine receptor desensitization, supporting a role for extracellular calcium in the homologous H1 receptor desensitization process.
Histamine (30 μm) stimulated at least four different types of [Ca2+]i signals in GT1‐7 cells. The majority (61%) were of single spikes with the remaining cells showing some form of calcium oscillatory behaviour. The proportion of GT1‐7 cells showing histamine‐induced calcium oscillations was histamine concentration‐dependent and significantly reduced after acute desensitization. KN‐62, when present during histamine pretreatment, prevented this fall in calcium oscillation. Under the conditions of nominal or 0.3 mM extracellular calcium the proportion of cells exhibiting histamine‐stimulated calcium oscillations was not significantly different from the controls.
Bradykinin stimulated a [Ca2+]i transient in GT1‐7 cells with a population mean peak response of 147 ± 8 nM (n = 5) over basal levels. The bradykinin‐induced [Ca2+]i signal was without any calcium oscillatory activity. Histamine pretreatment caused the heterologous desensitization of the bradykinin [Ca2+]i signal (44% reduction, P < 0.007), which was unaffected by KN‐62.
The results presented here suggest that the histamine‐mediated homologous H1 receptor desensitization process involves extracellular calcium and can be blocked by KN‐62, a putative inhibitor of CaMKII. In contrast, KN‐62 does not appear to prevent the histamine‐mediated heterologous desensitization cascade. These findings suggest fundamental differences in the mechanisms underlying homologous and heterologous H1 receptor desensitization pathways in GT1‐7 neuronal cells.
DOI: 10.1111/j.1476-5381.1996.tb15514.x
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