Large changes in intracellular pH and calcium observed during heat shock are not responsible for the induction of heat shock proteins in Drosophila melanogaster.

Abstract

Heat shock caused significant changes in intracellular pH (pH1) and intracellular free calcium concentration ([Ca2 k) which occurred rapidly after temperature elevation. pH1 fell from a resting level value at 25°C of 7.38 ± 0.02 (mean ± standard error of the mean, n = 15) to 6.91 ± 0.11 (n = 7) at 35°C. The resting level value of [Ca211, in single Drosophila melanogaster larval salivary gland cells was 198 ± 31 nM (n = 4). It increased approximately 10-fold, to 1,870 ± 770 nM (n = 4), during a heat shock. When salivary glands were incubated in calcium-free, ethylene glycol-bis(fO-aminoethyl ether)-N,N',N'-tetraacetic acid (EGTA)-buffered medium, the resting level value of [Ca2+]j was reduced to 80 ± 7 nM (n = 3), and heat shock resulted in a fourfold increase in [Ca2+1J to 353 4-90 nM (n = 3). The intracellular free-ion concentrations of Na+, K+, ClP, and Mg2' were 9.6 ± 0.8, 101.9 + 1.7, 36 + 1.5, and 2.4 + 0.2 mM, respectively, and remained essentially unchanged during a heat shock. Procedures were devised to mimic or block the effects of heat shock on pH, and[Ca2+]i and to assess their role in the induction of heat shock proteins. We report here that the changes in [Ca2+]i and pH; which occur during heat shock are not sufficient, nor are they required, for a complete induction of the heat shock response.Heating Drosophila melanogaster tissues or cultured cells from 25 to 35°C results in the rapid activation of heat shock genes and the selective translation of heat shock mRNA into protein, whereas all other transcription and translation are abruptly halted (1). This response to heat is a nearly universal phenomenon, occurring in procaryotes and in most eucaryotic cells. In addition, the heat shock proteins themselves and the mechanism of their induction have been highly conserved during evolution (1). These features have made the heat shock response one of the most widely used model systems for the study of eucaryotic gene expression.Heat shock genes can be activated by a wide variety of chemical agents, including oxidizing agents, heavy metals, ionophores, and amino acid analogs (1). A central question is whether these agents act in separate ways or whether they act on a common pathway for the expression of heat shock genes. The findings that polytene chromosome puffing at heat shock gene loci can occur within minutes after heating and that synthesis of heat shock mRNA is not affected by protein synthesis inhibitors both suggest that the induction pathway involves preexisting proteins which are activated by some physiological stimulus during a heat shock.Several lines of evidence suggest that the physiological stimulus may be provided by ionic changes in the cytoplasm of heat-shocked cells. Most notable is that treating cells with the ionophores valinomycin, dinactin, A23187, or ionomycin will result in heat shock protein synthesis (1, 25). Changes in plasma membrane fluidity, alterations in permeability to small molecules, and loss of membrane-bound enzyme activities have been shown to occur during heat shoc...

Mode of Action of aDrosophilaFMRFamide in Inducing Muscle Contraction

“…The reason for such potentiation is not clear but probably involves an increase in intracellular calcium levels in the muscle fibres. Warming Drosophila larval salivary gland cells to 35°C increases intracellular Ca 2+ concentration 10-fold, with a slow recovery of [Ca 2+ ] i , starting ~45 min after the temperature had cooled to 25°C (Drummond et al, 1986). If heat shock has a similar effect in muscle cells, an increase in intracellular Ca 2+ might last long enough to augment the response to the peptide.…”

Heat shock proteins: the search for functions.

“…Given this caveat, one finds a potpourri of data-too numerous to detail here-describing changes occurring in the heat-shocked cell (many are noted in references 13, 42, and 68). Among these are the cessation of DNA synthesis and condensation of chromatin (61), the dephosphorylation of ribosomal protein $6 (26,56,67), collapse of the intermediate filament network (11,75), increases in intraeellular calcium (18), shifts to glycolytic metabolism (28), etc. With such a broad assortment, it will be difficult to pinpoint where an HSP will function and an HSP70, for example, might have several sites for activity.…”

Intracellular pH measurements using flow cytometry with 1,4‐diacetoxy‐2,3‐dicyanobenzene

“…Modulation of pHi by 0.1 to 0.2 pH units can activate or suppress vital biochemical pathways such as glycolysis, gluconeogenesis, and urea and protein synthesis (4,5,13,14). Alterations in pH; can be accomplished through growth factors, extracellular pH (pH,) variations, proton ionophores, and weak acids/ weak bases (7,16,19).…”