Receptor-activated Ca2+ influx was investigated in PC12 cells clones loaded with fura-2. Cells were stimulated in a Ca2+-free medium and studied after reintroduction of the cation or addition of Mn2+ into the medium. A first influx component, independent of receptor activation and sustained by depletion of the intracellular inositol 1,4,5-trisphosphate sensitive Ca2+ store (store-dependent Ca2+ influx, SDCI), was identified by experiments with carbachol followed by atropine and with agents that induce store discharge without polyphosphoinositide hydrolysis: thapsigargin, an inhibitor of Ca2+-ATPase activity; ryanodine and caffeine, activators of the ryanodine receptor. A second component of Ca2+ influx, induced by carbachol and rapidly blocked by atropine, relies on receptor-effector coupling via G protein(s) different from that (those) involved in phospholipase C activation. SDCI and receptor-coupled influx are similar in their voltage dependence and insensitivity to forskolin and phorbol esters but they differ with respect to their Mn2+ permeability and their sensitivity to the SC 38249 imidazole blocker. The two components might play different roles. SDCI might act as a safety device to prevent Ca2+ store depletion whereas receptor-dependent influx might control physiological functions such as secretion and growth.

Alpha latrotoxin, purified from the venom of the black widow spider, is a high Mr (130000) protein devoid of detectable enzymatic activity. When applied to vertebrate nerve terminals (of the central as well as peripheral nervous systems) the toxin elicits massive release of neurotransmitters by stimulating the fusion of synaptic vesicles with the presynaptic membrane (exocytosis). Among non-neuronal systems, only the neurosecretory cell line PC12 is sensitive to alpha latrotoxin; all others investigated so far are insensitive. In order to act, alpha latrotoxin requires the presence of divalent cations in the medium. Ca2+ can be substituted by other divalent cations as Sr2+, Ba2+, Mn2+, Mg2+. However, with the last two the catecholamine release response is reduced in PC12 cells and synaptosomes. A specific, high affinity receptor of alpha latrotoxin exists in preparation sensitive to the toxin. This receptor has been purified and found to be a high Mr, integral membrane protein. In the frog neuromuscular junction the receptor is localized exclusively in the presynaptic membrane. Binding of alpha latrotoxin to the receptor in a Ca2+-containing incubation medium induces membrane depolarization (insensitive to tetrodotoxin), stimulation of Ca2+ influx (insensitive to verapamil) with consequent increase in the cytoplasmic free Ca2+ concentration and stimulation of phosphoinositide breakdown. In Ca2+ free medium depolarization is maintained, but free Ca2+ concentration does not rise after toxin application. In conclusion, alpha latrotoxin seems to act through a dual mechanism. The Ca2+-independent part of this mechanism may be mediated by the activation of protein kinase C, triggered by phosphoinositide metabolism. The relevance of these findings for presynaptic physiology is discussed.

The existence of a direct link between receptor-activated phospholipid hydrolysis and changes of cellular Ca2+ homeostasis was hypothesized quite some time ago but remained unclear, being based largely on anecdotal evidence, until the discovery almost a decade ago of the specific Ca2+ release activity of inositol 1,4,5-trisphosphate (IP3) (reviewed by Berridge and Irvine, 1989). Initially, the reaction responsible for IP3 generation, i.e., the hydrolysis of phosphatidyl inositol 4,5-bisphosphate (PIP2) by specific phospholipase(s) C (PLC), was thought to be the only metabolic process by which [Ca2+]i was regulated. However, the field soon became more complex. In 1984–85, activation of the receptors coupled to PPI hydrolysis was shown to stimulate not only Ca2+ release from internal stores but also Ca2+ influx via channels in the plasmalemma, later named second messenger-operated channels (Meldolesi and Pozzan, 1987). The multiplicity and complex regulation (not only by second messengers, but also via G proteins and the physiological state of intracellular Ca2+ stores) of these channels is discussed elsewhere (Meldolesi et al., 1991).