A high-throughput screen for MscL channel activity and mutational phenotyping.

A novel fluorescence-based screen for bacterial mechanosensitive ion-channel activity has been developed. This assay is capable of clearly distinguishing the previously observed gain of function and loss of function phenotypes for the Escherichia coli mechanosensitive channel of large conductance (Ec-MscL). The method modifies Molecular Probes' Live/Dead BacLight bacterial viability assay to monitor MscL channel activity as a function of bacterial survival from osmotic downshock.

Comparing and contrasting Escherichia coli and Mycobacterium tuberculosis mechanosensitive channels (MscL). New gain of function mutations in the loop region.

Sequence analysis of 35 putative MscL homologues was used to develop an optimal alignment for Escherichia coli and Mycobacterium tuberculosis MscL and to place these homologues into sequence subfamilies. By using this alignment, previously identified E. coli MscL mutants that displayed severe and very severe gain of function phenotypes were mapped onto the M. tuberculosis MscL sequence. Not all of the resulting M. tuberculosis mutants displayed a gain of function phenotype; for instance, normal phenotypes were noted for mutations at Ala(20), the analogue of the highly sensitive Gly(22) site in E. coli. A previously unnoticed intersubunit hydrogen bond in the extracellular loop region of the M. tuberculosis MscL crystal structure has been analyzed. Cross-linkable residues were substituted for the residues involved in the hydrogen bond, and cross-linking studies indicated that these sites are spatially close under physiological conditions. In general, mutation at these positions results in a gain of function phenotype, which provides strong evidence for the importance of the loop region in MscL channel function. No analogue to this interesting interaction could be found in E. coli MscL by sequence alignment. Taken together, these results indicate that caution should be exercised in using the M. tuberculosis MscL crystal structure to analyze previous functional studies of E. coli MscL.

Luteinizing hormone-releasing hormone quantified in tissues and slice explant cultures of postnatal rat hypothalami.

LH-releasing hormone (LHRH) peptide from postnatal rat preoptic area (POA)/hypothalamic tissues in vivo and slice explant cultures maintained in vitro was quantitated using an enzyme-linked immunosorbant assay. Moreover, messenger RNA (mRNA) copy number was calculated in LHRH neurons maintained in culture using in situ hybridization histochemistry with autoradiographic film analysis. POA/hypothalami from postnatal day 5-6 pups averaged 1250 pg of LHRH, with approximately 28% of peptide residing within rostral tissues where most LHRH perikarya reside. Explant cultures maintained 18 days in vitro contained 30.4-92.0 pg/slice with a whole animal total of 244.8 pg. Considering cell numbers in vivo and in vitro, LHRH neurons in whole animal produce 1.0 pg of LHRH/cell, whereas those in culture average 2.0 pg/cell. Furthermore, LHRH mRNA copies/cell in organotypic culture was estimated conservatively at 1410 copies/cell, a relatively high number. This work shows that, compared with whole animal, cultures have substantial LHRH stores, indicating maturation of synthetic activity and/or formation of new terminals in vitro. High LHRH mRNA copy number also suggests a high rate of peptide biosynthesis. Our analysis, demonstrating the dynamic potential of LHRH neurons, suggests that subtle changes in LHRH mRNA expression in all cells or a subpopulation can dramatically alter the LHRH system biosynthetic capacity.

Neuronal dopamine subpopulations maintained in hypothalamic slice explant cultures exhibit distinct tyrosine hydroxylase mRNA turnover rates.

Changes in mRNA stability have been shown to regulate critical intracellular processes. In this investigation, we studied tyrosine hydroxylase (TH) mRNA turnover in functionally and anatomically distinct dopaminergic (DA) populations of the rat hypothalamus. To this end, long-term slice explant cultures from postnatal, preoptic area/hypothalami, containing three anatomically discrete DA populations, were generated and maintained under defined conditions. The organotypic cultures were treated with the transcription inhibitors 5,6-dichloro-1-D-ribofuranosylbenzimidazole or actinomycin D and processed for in situ hybridization histochemistry. Relative TH mRNA content per cell was quantitated. Single-cell analysis showed marked differences in basal TH mRNA turnover rates between DA neuronal populations. Anterior and midhypothalamic DA neurons exhibited half-time turnovers of 9-12 and 11-23 hr, respectively. In contrast, in the caudal hypothalamus, DA neurons of the arcuate nucleus had a significantly lower baseline level and more rapid turnover (6-7 hr) of TH mRNA. This investigation shows that basal turnover of a phenotypic mRNA, TH mRNA in DA neurons, is not an intrinsic property of the phenotypic marker. Furthermore, we found that destabilization of TH mRNA in the caudal hypothalamus corresponds to the known rhythmic output displayed by arcuate DA cells and, as such, may be critical for normal function of this population. We propose that intrinsic differences in the post-transcriptional regulation of TH permits neuronal subpopulations, which subserve different physiological functions, an additional mechanism to control DA biosynthesis in response to their unique needs.

Luteinizing hormone-releasing hormone (LHRH) neurons maintained in hypothalamic slice explant cultures exhibit a rapid LHRH mRNA turnover rate.

Evidence indicates that neuropeptide gene expression is tightly coupled to biosynthesis and secretion. Moreover, rhythmic gene expression often accompanies rhythmic secretion. Luteinizing hormone-releasing hormone (LHRH) neurosecretion, which regulates gonadal function, is pulsatile, with interpulse intervals of approximately 1 hr and pulse decays of <30 min in rats. As a basis for a rapid fall in peptide secretion, we hypothesize that LHRH mRNA levels rapidly decay. To address this hypothesis, we examined LHRH mRNA turnover in primary postnatal LHRH neurons maintained in long-term hypothalamic/preoptic area slice explant cultures, using in situ hybridization histochemistry (ISHH). Relative LHRH mRNA content per cell was quantitated by single-cell analysis after transcription inhibition with 5, 6-dichloro-1-D-ribofuranosyl-benzimidazole (DRB) or actinomycin D. Cultures were maintained in serum-free medium with tetrodotoxin to suppress spontaneous electrical activity and hence assess only intrinsic cellular activity. A plot of LHRH mRNA level per cell versus DRB treatment time showed a rapid initial decay of LHRH mRNA (t1/2, 5-13 min), followed by a slower decay rate (t1/2, 329-344 hr). LHRH cell number after drug treatment as determined by immunocytochemistry did not change. Comparison of mammalian LHRH mRNA 3'-untranslated regions showed two conserved regions. These data indicate that, in primary LHRH neurons, LHRH mRNA has an intrinsically high rate of turnover and a mRNA stabilization component. Foremost, decay of LHRH mRNA, the fastest reported for a neuropeptide to date, corresponds to the decay of LHRH peptide pulses.

Effects of protein kinase inhibitors on morphology and function of cultured bovine adrenal chromaffin cells: KN-62 inhibits secretory function by blocking stimulated Ca2+ entry.

In cultured bovine adrenal chromaffin cells, a nonselective protein kinase inhibitor, staurosporine, inhibits secretory function and induces neurite outgrowth. In the present study, effects of other nonselective protein kinase inhibitors (K-252a, H-7, and H-8) and reportedly selective protein kinase inhibitors (KN-62 and chelerythrine chloride) were examined on bovine adrenal chromaffin cell morphology, secretory function, and 45Ca2+ uptake. Treatment of chromaffin cells with 10 microM K-252a, 50 microM H-7, or 50 microM H-8 induced changes in cell morphology within 3 h; these compounds also induced a time-dependent inhibition of stimulated catecholamine release. Chelerythrine chloride, a selective inhibitor of Ca2+/phospholipid-dependent protein kinase, did not induce outgrowth or inhibit secretory function under our treatment conditions. KN-62, a selective inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMK II), significantly inhibited stimulated catecholamine release (IC50 value of 0.32 microM), but had no effect on cell morphology. The reduction of secretory function induced by 1 microM KN-62 was significant within 5 min and rapidly reversible. Unlike H-7, H-8, and staurosporine, KN-62 significantly inhibited stimulated 45Ca2+ uptake. KN-04, a structural analogue of KN-62 that does not inhibit CaMK II, inhibited stimulated 45Ca2+ uptake and catecholamine release like KN-62. These studies indicate that KN-62 inhibits secretory function via the direct blockade of activated Ca2+ influx. The nonselective inhibitors, K-252a, H-7, H-8, and staurosporine, inhibit secretory function by another mechanism, perhaps one involving alterations in the cytoskeleton.

Staurosporine affects calcium homeostasis in cultured bovine adrenal chromaffin cells.

These studies show that the potent, non-specific, protein kinase inhibitor, staurosporine, disrupts Ca2+ homeostasis in cultured bovine adrenal chromaffin cells. Staurosporine treatment reduces basal and A23187-stimulated catecholamine release from chromaffin cells, but does not inhibit activated Ca2+ influx. Furthermore, pretreatment with staurosporine also reduces Ca(2+)-stimulated catecholamine release from digitonin-permeabilized cells (t1/2, 40.6 min; IC50, 66.0 nm). However, staurosporine does not inhibit the rise in intracellular Ca2+ ([Ca2+]i) in response to nicotine stimulation as measured by fura-2 photometry. These studies demonstrate that staurosporine interferes with the secretory process at some step at or after the rise in [Ca2+]i in adrenal chromaffin cells. Examination of the effects of staurosporine on 45Ca2+ movement shows that staurosporine produces a slowly developing basal 45Ca2+ accumulation; after 30 min no significant change is observed, but by 120 min, 45Ca2+ accumulation is increased by 29.5%. Thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBHQ), inhibitors of Ca(2+) ATPases, were used to determine whether staurosporine induced 45Ca2+ accumulation results from sequestration of 45Ca2+ within intracellular stores. While thapsigargin has no significant effect, concomitant treatment with tBHQ prevents the increase in 45Ca2+ uptake associated with staurosporine treatment. Therefore, the tBHQ-sensitive Ca2+ store, but not the thapsigargin/inositol 1,4,5-triphosphate-sensitive Ca2+ store, appears to be staurosporine-sensitive. Overall, these studies indicate that staurosporine reduces catecholamine release by interfering with Ca2+ homeostasis. Furthermore, this work suggests that a staurosporine-sensitive phosphoprotein(s) is involved with the regulation of Ca2+ homeostasis in bovine adrenal chromaffin cells.

Staurosporine-induced reduction of secretory function in cultured bovine adrenal chromaffin cells.

Staurosporine, a potent inhibitor of protein kinases, has been used to investigate the involvement of protein kinases in cellular processes such as secretory function and differentiation. We have been examining the effects of staurosporine on secretory function under the same conditions it induces dramatic changes in cell morphology in cultured bovine adrenal chromaffin cells. Our results show that treatment with 100 nM staurosporine reduces catecholamine release stimulated by 56 mM KCl, 10 microM nicotine, and 2 mM BaCl2 in a time-dependent manner (t1/2s, 42, 32, and 31 min, respectively). However, we demonstrate that the time-dependent effects on secretory function are not the direct result of staurosporine-induced changes in cell morphology. The effects of staurosporine on secretion stimulated by KCl, nicotine, and BaCl2 are concentration-dependent (IC50s, 6.3, 29.3, and 34.9 nM, respectively). Staurosporine pretreatment does not inhibit activated 45Ca2+ influx, but does reduce catecholamine release stimulated directly by Ca2+ from permeabilized cells. Furthermore, staurosporine also inhibits basal release with time- and concentration-dependencies (IC50, 9.3 nM and t1/2, 21 min) similar to those found for stimulated release. These results suggest that prolonged staurosporine pretreatment may result in the depletion/alteration of a component essential for the more terminal steps of the secretory process.

Time-dependent inhibition of stimulated adrenal catecholamine release by staurosporine.

Staurosporine, a potent inhibitor of protein kinases, is used to study the involvement of protein kinases in cellular processes. In the present studies, the effect of prolonged staurosporine treatment on catecholamine secretion in cultured bovine adrenal chromaffin cells was examined. Staurosporine inhibits catecholamine release stimulated by 10 microM nicotine, depolarizing concentrations of potassium (56 mM KCl), and 2 mM BaCl2. The effects of staurosporine on KCl-stimulated release are time dependent, with a half-time of approximately 50 min and a maximal inhibition at 2 h. Our results indicate that activation of a staurosporine-sensitive protein kinase is not directly involved in the stimulus-secretion coupling process. This does not rule out the possibility that Ca2+/phospholipid-dependent protein kinase or other protein kinases may acutely modulate release. However, these results suggest that a protein(s), which is phosphorylated by a staurosporine-sensitive protein kinase(s), is required to maintain the integrity of the stimulus-secretion coupling process.