Human peripheral blood lymphocytes regulate their volumes in hypotonic solutions. In hypotonic media in which Na' is the predominant cation, an initial swelling phase is followed by a regulatory volume decrease (RVD) associated with a net loss of cellular K + . In media in which K+ is the predominant cation, the rapid initial swelling is followed by a slower second swelling phase . s6Rb+ fluxes increased during RVD and returned to normal when the original volume was approximately regained . Effects similar to those induced by hypotonic stress could also be produced by raising the intracellular Ca" level . In isotonic, Ca"-containing media cells were found to shrink upon addition of the Ca" ionophore A23187 in K+ -free media, but to swell in K + -rich media . Exposure to Ca" plus A23187 also increased s6Rb+ fluxes . Quinine (75 LM), an inhibitor of the Ca"-activated K+ pathway in other systems, blocked RVD, the associated K + loss, and the increase in s6Rb + efflux . Quinine also inhibited the volume changes and the increased 86 Rb fluxes induced by Ca" plus ionophore . The calmodulin inhibitors trifluoperazine, pimozide, and chlorpromazine blocked RVD as well as Ca" plus A23187-induced volume changes . Trifluoperazine also prevented the increase in s6Rb+ fluxes and K+ loss induced by hypotonicity . Chlorpromazine sulfoxide, a relatively ineffective calmodulin antagonist, was considerably less potent as an inhibitor of RVD than chlorpromazine . It is suggested that an elevation in cytoplasmic [Ca"], triggered by cell swelling, increases the plasma membrane permeability to K + , the ensuing increased efflux of K+ , associated anions, and osmotically obliged water, leading to cell shrinking (RVD) .

Peripheral blood mononuclear cells (PBM) readjust their volumes after swelling in hypotonic media. This regulatory volume decrease (RVD) is associated with a loss of cellular K + and is thought to be promoted by an increased permeability to this ion. In contrast, no change in volume was observed when K + permeability of PBM in isotonic media was increased to comparable or higher levels using valinomycin. Moreover, valinomycin-induced 86Rb+ loss in K+-free medium was considerably slower than in K+-rich medium. These results suggest that anion conductance limits net salt loss in isotonic media. Direct measurements of relative conductance confirmed that in volume-static cells, anion conductance is lower than that of K +. In volume-regulating cells depolarization occurred presumably as a result of increased anion conductance. Accordingly, the effiux of a6CI from PBM was markedly increased by hypotonic stress. Since both membrane potential and intracellular ant1 concentration are reduced in hypotonically swollen cells, the increased efflux is probably due to a change in CI-permeability. Anions and cations seem to move independently through the volume-induced pathways: the initial rate ofsnRb uptake in swollen cells was not affected by replacement of external C1-by SO7; conversely, 36C1 fluxes were unaffected by substitution of K + by Na +. The data indicate that anion conductance is rate-determining in salt and water loss from PBM. An increase in anion conductance is suggested to be the critical step of RVD of human PBM.

Phenylalkylamines such as 1-(4-bromo-2, 5-dimethoxyphenyl)-2-aminopropane (DOB; 1a) and its corresponding iodo derivative DOI (2) are commonly used 5-HT(2) serotonin agonists. Previous studies have established that the 2,5-dimethoxy substitution pattern found in these compounds is optimal for high affinity at 5-HT(2A) receptors and that substituents at the 4-position can modulate affinity over a wide range. We have previously shown, however, that when the 4-position is substituted with a 3-phenylpropyl substituent (i.e., 3), the compound binds with an affinity comparable to that of 1a but that it possesses 5-HT(2A) antagonist character. The present study examined the structure-affinity relationships of 3, and the results were very much unexpected. That is, the 2,5-dimethoxy substitution pattern of 3 is not required for high affinity. Either of the two methoxy groups can be removed without untoward effect on affinity, and relocation of the methoxy substituents actually enhances affinity by as much as an order of magnitude. None of the compounds displayed more than 20-fold selectivity for 5-HT(2A) over 5-HT(2C) receptors. In addition, several were demonstrated to act as 5-HT(2A) partial agonists. As such, the results of this study suggest that the structure-affinity relationships of phenylalkylamines as 5-HT(2A) ligands now be reinvestigated in greater detail.

The ternary complex model as applied to G-protein coupled receptors (GPCR) predicts that an agonist binds with low affinity (K(L)) to the free receptor (R), leading to an agonist/receptor/G-protein complex. This ternary complex displays high agonist affinity (K(H)), resulting in signal transduction. Classical dogma states that the ratio K(L)/K(H) predicts intrinsic activity of drugs: the higher the ratio the higher the intrinsic activity. This model was based on studies in which K(L) and K(H) were indirectly determined by computer analyses of antagonist radioligand binding data. In order to investigate the relationship of K(L), K(H), and intrinsic activity for agonists at 5-HT(2A) and 5-HT(2C) receptors, we utilized (3)H-agonist and (3)H-antagonist radioligands to directly determine K(H) and K(L). Comparisons of the log K(L)/K(H) ratios and intrinsic activities of drugs for stimulating intracellular phosphatidylinositol (PI) hydrolysis revealed a strong correlation for 5-HT(2A) (r(2) = 0.92) and 5-HT(2C) (r(2) = 0.96) receptors. The data were fit to computer simulations based on the original ternary complex model and the revised ternary complex model in which an activated state of the receptor (R*) exists in equilibrium with the resting state of the receptor (R). Data produced for both 5-HT(2A) and 5-HT(2C) receptors were better-fitted to a revised ternary complex model, rather than the classical ternary complex model. These data support a revised model for the molecular events coupling GPCR to activation of G-proteins and indicate that a strong correlation between the K(L)/K(H) ratio and intrinsic activity for agonist action at GPCR is consistent with the existence of R*.