Sphingosine interacts directly with the receptor complex to inhibit thyrotropin-releasing hormone binding.

Sphingosine inhibition of [3H] [N3-Me-His] TRH (MeTRH) binding, previously shown to be independent of its effects on protein kinase-C, has been further characterized in GH3 cell membranes and in a partially purified, digitonin-solubilized receptor preparation. In membranes, as in intact cells, sphingosine inhibited [3H]MeTRH binding by decreasing receptor affinity, but, in contrast to its effect in intact cells, did not affect the number of available binding sites. The inhibition of binding was linear up to 75 microM sphingosine (in the presence of 100 microM BSA at 0.1 mg membrane protein/ml), yielding an apparent Ki of 51 microM. Since GTP decreases the affinity for MeTRH binding in GH3 cell membranes, we studied interactions between GTP and sphingosine. While the effects of low concentrations of GTP gamma S and sphingosine were additive, sphingosine inhibition of MeTRH binding surpassed and was not affected by the addition of maximally inhibitory concentrations of GTP gamma S. Also, sphingosine (75 microM) did not affect the ability of a maximally effective dose of TRH to stimulate the low Km GTPase (vehicle, +35 +/- 5%; sphingosine, +32 +/- 10%); there was a 25% decrease in total GTPase activity in the presence of sphingosine. MeTRH binding to digitonin-solubilized receptors, which had properties similar to those described previously by others, including no effect of GTP on binding, was inhibited by sphingosine. In solubilized receptors, as in membranes, sphingosine caused a decrease in apparent affinity without changes in the number of binding sites. These data suggest that sphingosine interacts directly with the TRH receptor [or an associated factor(s) in the receptor complex] to decrease affinity by a mechanism that does not involve uncoupling of G-proteins.

Complex interactions of agonists with alpha 1-adrenoceptors in intact cells.

1. The apparent Ki values of (-)-noradrenaline (NA), (+)- and (-)-adrenaline (Ad), phenylephrine and the mono-fluorinated NAs (in position 2, 5 or 6) for alpha 1-adrenoceptors of intact BC3H1 cells labelled with [3H]-prazosin were greatly dependent on the incubation temperature. 2. The EC50 values of these compounds for stimulation of the inositol phosphate (IP) accumulation at 37 degrees C were intermediate between their apparent dissociation constants at 2 degrees C (Ki2 degrees) and at 37 degrees C (Ki37 degrees). 3. The fact that an irreversible blockade of 46% +/- 6% (n = 3) of the [3H]-prazosin binding sites by phenoxybenzamine reduced the maximal IP-formation induced by NA by 57% +/- 5% (n = 3) shows that there is a direct coupling between alpha 1-adrenoceptors and phospholipase C in BC3H1 cells. 4. The Ki37 degrees s of all agonists tested were in the same range (0.1 to 1 mM) and showed no simple correlation with their EC50 values. 5. The Ki2 degrees values for all the agonist correlated linearly with their EC50 values but were about 20-100 times lower than the respective EC50 values (except for the partial agonist methoxamine). In order to explain this difference, we propose that the apparent high affinity in the cold could be due to an [3H]-prazosin-induced alteration of the active site of the alpha 1-adrenoceptor, increasing its apparent affinity for catecholamines.

New insights into maitotoxin action.

Maitotoxin (3 ng/mol) induced a massive uptake of 45Ca2+ into BC3H1 cells. This effect exhibits a lag phase of 3 min. Inositol diphosphate formation occurred concomittantly with the 45Ca2+ uptake but inositol monophosphate formation was found only after a 5-min delay following toxin addition. Maitotoxin-induced 45Ca2+ influxes could not be blocked by either 1 microM verapamil, 1 microM nifedipine or 1 mM La3+ but was blocked by Zn2+ (IC50 = 41 microM). In addition to inositol phosphate formation and 45Ca2+ uptake, maitotoxin stimulated a large uptake of Na+ and a great loss of K+ in BC3H1 cells. In the absence of Ca2+ (1 mM EGTA) none of the four maitotoxin effects could be detected. After restoration of Ca2+, the maitotoxin effects reappeared even when the toxin itself was no longer present. The divalent cation, Co2+ (1 mM), inhibited ion movements induced by maitotoxin and also digitonin (8.1 microM). The toxin action showed a very pronounced pH dependence. At low pH, maitotoxin was inactive. The dose-response curves for H+ ion inhibition of maitotoxin-induced Ca2+ uptake showed a shift to the right when determined in the absence of HCO3- and HCO3-/Cl- ions. It was concluded that the primary action of maitotoxin in BC3H1 cells was a pore-forming or channel-forming activity of a non-classical type. Some properties of maitotoxin resemble those of alpha-latrotoxin, others those of pore-forming agents such as melittin or alpha-toxin of Staphylococcus aureus.

Piperazine derivatives including the putative anxiolytic drugs, buspirone and ipsapirone, are agonists at 5-HT1A receptors negatively coupled with adenylate cyclase in hippocampal neurons.

Two putative anxiolytic drugs [ipsapirone (TVXQ 7821) and buspirone], structurally unrelated to benzodiazepines, have negligible ataxic and sedative side effects. These drugs are piperazine analogs which interact at 5-HT1 binding sites. It is demonstrated here that these drugs and two other piperazine derivatives, trifluoromethylphenylpiperazine (TFMPP) and m-chlorophenylpiperazine (mCPP), are agonists at 5-HT1A receptors, a subclass of the 5-HT1 receptor, mediating inhibition of forskolin (100 microM) stimulated adenylate cyclase in particulate fractions of guinea pig hippocampus as well as inhibition of the formation of cyclic AMP promoted by vasoactive intestinal polypeptide (0.1 microM) plus forskolin (1 microM) in mouse hippocampal neurons in primary culture. This study demonstrates that these piperazine based drugs act in both brain homogenate preparations and in intact neurons in a similar manner. The biochemical models described here may aid in the development of even more active drugs in this class.

The 5-hydroxytryptamine (5-HT2) receptor stimulates inositol phosphate formation in intact and broken WRK1 cells: determination of occupancy-response relationships for 5-HT agonists.

5-Hydroxytryptamine (5-HT) stimulates the accumulation of inositol-trisphosphate in WRK1 cells, a cell line originating from a rat mammary tumor. 5-HT acts via a single receptor type for which it has an affinity constant estimated to be 1.27 microM. A series of agonists known to act at 5-HT2 receptors are partial agonists in this system and have a rank order of relative intrinsic efficacies corresponding to that seen in other systems possessing 5-HT2 receptors. There is an essentially linear occupancy-response relationship for 5-HT and other agonists indicating the absence of a strong amplification mechanism between receptor activation and inositol phosphate formation. The selective blockade of the 5-HT response by nanomolar concentrations of 5-HT2 selective antagonists but not by drugs acting at other 5-HT receptor subtypes suggest that the receptor in WRK1 cells is of the 5-HT2 type. Additionally, we demonstrate that in WRK1 membranes 5-HT acts via the 5-HT2 receptor to elicit a GTP dependent increase in the production of inositol-bisphosphate and inositol-trisphosphate. These properties of the WRK1 cell line indicate that it is a useful model with which to study the nature of 5-HT receptor coupling to the putative second messenger(s), the inositol phosphates.

5-HT2 receptor-stimulated inositol phosphate formation in rat aortic myocytes.

Serotonin (5-HT) stimulated inositol phosphate production in primary cultures of rat aortic myocytes via a 5-HT2 receptor. Agonists active at 5-HT2 receptors in other systems were also active here but the response to some agonists was potentiated by the hormone uptake blocker, cocaine HCl. Two 5-HT2 selective antagonists, ketanserin and spiperone, inhibited the serotonin-induced response while compounds selective for other 5-HT receptor subtypes did not.

Kinetic definition of agonist efficacy at a 5-hydroxytryptamine (5-HT2) receptor in the isolated rabbit aorta.

The contractile response of the isolated rabbit aorta elicited by 5-hydroxytryptamine (5-HT) and five partial agonists acting on the 5-HT2 receptor were separated into a phasic and a tonic response by altering the [Ca++] in the buffer. A kinetic analysis of the two responses yields parameters that provide a mechanistic insight into the different nature of these responses. The kinetic parameters of the phasic contraction indicate that the onset of this response depends on the access of the drug to the receptor and that its decay is independent of the nature and the concentration of the agonist. The observed rate constant of the onset of the tonic response, kobs, is saturable with increasing drug concentration, suggesting that the rate determining step is the activation of an effector by the preformed drug-receptor complex. These kinetic characteristics of the 5-HT2-mediated response are similar to those observed previously by us for the alpha-1 adrenergic receptor-mediated response in the rabbit aorta, suggesting that these receptors activate similar mechanisms related to the mobilization of Ca++. Furthermore, it is shown that the maximal values of kobs for the 5-HT2 agonists follow the rank order of maximal amplitudes of the phasic responses and the maximal steady-state levels of the tonic response. It is suggested that the maximal value of kobs may serve as a kinetic measure of drug efficacy.

Kinetic characterization of the rabbit aorta contractile response to an alpha adrenergic agonist.

The alpha-1 adrenergic response of the rabbit aorta to phenylephrine (PE) was separated into a phasic and a tonic response by virtue of their different dependence on extracellular [Ca++]. The kinetics of each response was characterized with respect to its dependence on [PE] and [Ca++]. The phasic response is independent of extracellular calcium and has a rapid onset followed by a first order decay. Although its maximal attainable response is saturable with respect to [PE] and [Ca++], its rate constant for onset does not depend on the concentration of calcium in the preincubation buffer. We were unable to show that this rate constant for onset is saturable with respect to [PE]. This suggests that the rate-determining step of the phasic response is the diffusion-controlled formation of the drug-receptor complex. The tonic response depends on extracellular calcium, shows first order kinetics of onset and reaches a steady-state level of contraction that is saturable with respect to [PE] and extracellular [Ca++]. The rate constant for the generation of the tonic response depends on [PE] in a saturable manner and linearly on extracellular [Ca++]. This suggests that the rate-determining step could be the activation of a hypothetical effector by the drug-receptor complex. The activated effector would enable the transport of calcium ions into the cell. The kinetic studies predict that the efficacy of a drug in this system is the maximal rate of activation of the effector by the drug-receptor complex.