Signals mediated by heterotrimeric G proteins often develop over the course of tens of milliseconds, and could require either conformational rearrangement or complete physical dissociation of Gα βγ heterotrimers. Although it is known that some active heterotrimers are dissociated (into Gα and Gβγ) at steady-state, it is not clear that dissociation occurs quickly enough to participate in rapid signaling. Here we show that fusion proteins containing the c-terminus of GPCR kinase 3 (GRK3ct) and either the fluorescent protein cerulean or Renilla luciferase bind to venus-labeled Gβγ dimers (Gβγ-V), resulting in Förster or bioluminescence resonance energy transfer (FRET or BRET). GRK3ct fusion proteins are freely-diffusible, and do not form preassembled complexes with G proteins. GRK3ct fusion proteins bind to free Gβγ-V dimers but not to rearranged heterotrimers, and thus can report G protein dissociation with high temporal resolution. We find that heterotrimer dissociation can occur in living cells in less than 100 milliseconds. Under the conditions of these experiments diffusion and collision of masGRK3ct fusion proteins and Gβγ-V were not rate-limiting. These results indicate that G protein heterotrimers can dissociate quickly enough to participate in rapid signaling.

Genes expressed specifically in a tissue are often involved in the defining functions of that tissue. We used representational difference analysis of cDNA to amplify 20 cDNA fragments representing transcripts that were more abundant in the lobster olfactory organ than in brain, eye/eyestalk, dactyl, pereiopod, or second antenna. We then independently confirmed that the transcripts represented by these clones were enriched in the olfactory organ. The 20 cDNA fragments represent between 6 and 15 different genes. Six of the cDNAs contained sequences highly similar to known gene families. We performed in situ hybridization with these six and found that all were expressed in subsets of cells associated with the aesthetasc sensilla in the olfactory organ. Clones OET-07, an ionotropic receptor, and OET-10, an alpha tubulin, were specific to the olfactory receptor neurons. OET-02, a monooxygenase, was expressed only in the outer auxiliary cells. OET-03, a serine protease, was specific to the collar cells. OET-11, an alpha(2) macroglobulin, was expressed by the receptor neurons and the collar cells. OET-17, a calcyphosine, was expressed in the receptor neurons, inner auxiliary cells, and collar cells. The identities and expression patterns of these six transcripts predict involvement in both known and novel properties of the lobster olfactory organ.

1. Current- and voltage-clamp studies were conducted on isolated rat adrenal chromaffin cells to identify the voltage-dependent ion channels mediating inward currents. 2. Mean resting membrane potential of the isolated cells was -62 +/- 3 (SE) mV. Evoked action potentials were both Na+ and Ca2+ based, and whole cell voltage-clamp studies in normal saline revealed an inward-rectifier-type current. 3. Na+ channels were studied in isolation and showed a half-inactivation of -60 +/- 2 mV with a slope factor of -6 mV and a half-activation of -26.8 +/- 2 mV with a slope factor of 6.5 +/- 0.7 mV. 4. Isolated Ca2+ currents, elicited in 10 mM external Ca2+, revealed a T-type current in a subset of cells. Ca2+ currents were sensitive to both N- and L-type channel antagonists, and blockade of the current by the L-type channel antagonist nimodipine and the N-type channel antagonist omega-conotoxin GVIA revealed a third Ca2+-current component that was unaffected by the P-type channel antagonist omega-agatoxin IVA. 5. Ca2+ currents were facilitated 5-20% by a depolarizing prepulse, and facilitation was completely blocked by nimodipine. The effects of the dihydropyridine L-type channel agonist, (+)202-791 and depolarizing prepulses on the currents were additive. 6. The results of this study show that the properties of voltage-dependent ion channels in rat chromaffin cells differ from those reported in their counterparts in bovine chromaffin cells. Na+ channels differ in activation and inactivation properties and Ca2+ channels differ in activation, sensitivity to antagonists, and the magnitude of voltage-dependent facilitation.

A cloned P2x-purinoceptor was transiently expressed in single isolated rat adrenal chromaffin cells and evaluated for the detection of released ATP. After cytoplasmic injection of the P2x complementary RNA (cRNA; 4-24 h), application of ATP produced an inwardly rectifying current over the voltage range -130 to -10 mV as measured by the whole cell patch-clamp technique. The dose-response curve for ATP was sigmoidal with a 50% effective concentration of 18. 2 microM. Suramin, a P2x-antagonist, attenuated the ATP-induced current. Depolarizing voltage pulses to 0 mV or application of histamine, stimuli that trigger exocytosis, resulted in the appearance of suramin-sensitive spontaneous transient inward currents (at -60 mV) that resembled excitatory postsynaptic currents although they were slower in time course. Concurrent detection of catecholamine release with a carbon fiber electrode often showed coincidence of the amperometric current with the synaptic currentlike events suggesting that ATP and catecholamines were released from the same vessicle. These data demonstrate that expression of a P2x-purinoceptor in chromaffin cells produces a functional autoreceptor capable of detecting vesicular release of ATP. In combination with carbon fiber amperometry, simultaneous vesicular release of two neurotransmitters from a single chromaffin cell could be monitored. The P2x-purinoceptor, however, produced a regenerative effect on release apparently resulting from the high Ca2+ permeability of the receptor. Thus modification of the P2x-purinoceptor would be required before the system could be applied to examining processes involved in stimulus-release coupling.

Lobster olfactory sensory neurons have contributed to a number of advances in our understanding of olfactory physiology. To facilitate further study of their function, we have developed conditions allowing primary culture of the olfactory sensory neurons in a defined medium. The most common cells in the culture were round cell bodies with diameters of 10-15 micro m that often extended fine processes, features resembling olfactory sensory neurons. We discovered that acetylcholinesterase acted as a growth factor for these cells, improving their survival in culture. We also confirmed previous evidence from spiny lobsters that poly-D-lysine was a superior substrate for olfactory cells of this size and morphology. We then identified olfactory sensory neurons in the culture in two ways. Almost half the cells tested responded to application of a complex odorant with an inward current. An even more rigorous test was made possible by the development of an antiserum to OET-07, an ionotropic glutamate receptor homolog specifically expressed by Homarus americanus olfactory sensory neurons. It labeled a majority of the round cells in the culture, unequivocally identifying them as olfactory sensory neurons.

Three types of alpha-melanocyte-stimulating hormone (alpha MSH) that differ in the acetyl status of the N-terminal serine have been found in the neurointermediate lobe of the pituitary gland and in the brain: desacetyl alpha MSH, which lacks an acetyl group; monoacetyl alpha MSH, in which the amino group of the serine is acetylated; and diacetyl alpha MSH, in which both amino and hydroxy groups of the serine are acetylated. We compared the lipolytic and melanotropic actions of these three peptides, and their rates of disappearance from plasma, both in vitro and in vivo. The following differences were found. a) For in vitro lipolytic actions on rabbit adipose tissue slices, the potencies differed according to the order diacetyl = monoacetyl greater than desacetyl. On rabbit isolated adipocytes, however, the three peptides were equipotent. b) For in vivo lipolytic action in the rabbit, not only potency but also kinetics differed. Diacetyl alpha MSH had the slowest onset, longest duration, and greatest potency. The desacetyl variant had the quickest onset, shortest duration, and least potency. c) The half-life for elimination from rabbit plasma both in vitro and in vivo was shortest for the desacetyl form and longest for the diacetyl peptide. d) For in vitro melanotropic effect on frog skin, kinetics of action were the same for all three peptides, but potency differed according to the order diacetyl = monoacetyl greater than desacetyl. Thus acetylation of alpha MSH alters lipolytic and melanotropic potencies in vitro and lipolytic potency and kinetics in vivo. These differences result in part from the fact that acetylation slows the degradation of the tridecapeptide both inside and outside the circulation.

We have isolated from an American lobster (Homarus americanus) olfactory organ cDNA library a clone, lobGαs, with >70% identity to mammalian and arthropod Gαs sequences. In genomic Southern blots, a fragment of lobGαs detected only one band, suggesting the lobsters have a single Gαs gene. In brain and olfactory organ, lobGαs mRNA was expressed predominantly in neurons, including many of the neuronal cell body clusters of the brain. Gαs protein was also expressed broadly, appearing on western blots as a band of 51.8 kDa in brain, eyestalk, pereiopod, dactyl, tail muscle, olfactory organ, and aesthetasc hairs. These results suggest that lobGαs plays a role in a wide variety of signal transduction events. Its presence in the olfactory aesthetasc hairs, which are almost pure preparations of the outer dendrites of the olfactory receptor neurons, and the expression of lobGαs mRNA in the olfactory receptor neurons of the olfactory organ indicate that lobGαs may mediate olfactory transduction. That virtually all ORNs express lobGαs mRNA equally predicts that hyperpolarizing odor responses mediated by cyclic AMP are a property of all lobster olfactory receptor neurons.

Choroid plexus of rabbit and rat was incubated for 2-30 min at 37 degrees C under 95% O2-5% CO2 in Tyrode solution containing 10 mM glucose and 1 mM theophylline with these agents: epinephrine, norepinephrine, isoproterenol, dopamine, histamine, serotonin, arginine, and lysine vasopressins, oxytocin, angiotensin, adrenocorticotropin (ACTH), beta-melanocyte-stimulating hormone, and choroid plexus peptide IIF. After incubation, tissue and medium were analyzed for 3', 5' -cyclic adenosine monophosphate (cAMP) content. Each amine or peptide was tested initially at 1,000 microng/ml. Only ACTH and serotonin affected cAMP content of rabbit choroid plexus. At 1,000 microng/ml, these agents caused a 10 and 4 times (respectively) increase in cAMP content of tissue + medium at 2-10 min with decline in content at 10-30 min. More than 90% of the increment was located in tissue, less than 10% in medium. Minimal effective dose (MED) to cause a significant (P less than .05) accumulation of cAMP was 0.1 microng/ml (2.2 x 10(-8) M) for ACTH and 10 microng/ml (5.7 x10(-3) M) for serotonin. Only isoproterenol, epinephrine, and norepinephrine influenced cAMP content of rat choroid plexus. MED's for this effect by isoproterenol, epinephrine, and norepinephrine were .001, .01, and 10 microng/ml (4.7 x 10(-9), 5.5 x 10(-8), and 5.9 x 10(-5) M), respectively.