Zvi Selinger

Highly selective agonists for substance P receptor subtypes

The existence of a third tachykinin receptor (SP‐N) in the mammalian nervous system was demonstrated by development of highly selective agonists. Systematic N‐methylation of individual peptide bonds in the C‐terminal hexapeptide of substance P gave rise to agonists which specifically act on different receptor subtypes. The most selective analog of this series, succinyl‐[Asp6,Me‐Phe8]SP6‐11, elicits half‐maximal contraction of the guinea pig ileum through the neuronal SP‐N receptor at a concentration of 0.5 nM. At least 60,000‐fold higher concentrations of this peptide are required to stimulate the other two tachykinin receptors (SP‐P and SP‐E). The action of selective SP‐N agonists in the guinea pig ileum is antagonized by opioid peptides, suggesting a functional counteraction between opiate and SP‐N receptors. These results indicate that the tachykinin receptors are distinct entities which may mediate different physiological functions.

Colonic substance P levels are increased in ulcerative colitis and decreased in chronic severe constipation

Substance P content was determined by radioimmunoassay in colonic mucosa from 24 patients with chronic severe constipation, 16 with active ulcerative colitis, and 28 normal controls. In patients with chronic severe constipation, the mean concentration of substance P (19.9±8.2 pg/mg) was significantly lower than in normal subjects (71±18 pg/mg). In patients with ulcerative colitis, colonic substance P concentration in inflamed mucosa (170±46 pg/mg) was significantly higher than its levels in normal subjects. Substance P may therefore have a role in the pathogenesis of clinical conditions associated with diarrhea and constipation.

Normal Phototransduction in Drosophila Photoreceptors Lacking an InsP3 Receptor Gene

The Drosophila light-sensitive channels TRP and TRPL are prototypical members of an ion channel family responsible for a variety of receptor-mediated Ca(2+) influx phenomena, including store-operated calcium influx. While phospholipase Cbeta is essential, downstream events leading to TRP and TRPL activation remain unclear. We investigated the role of the InsP(3) receptor (InsP(3)R) by generating mosaic eyes homozygous for a deficiency of the only known InsP(3)R gene in Drosophila. Absence of gene product was confirmed by RT-PCR, Western analysis, and immunocytochemistry. Mutant photoreceptors underwent late onset retinal degeneration; however, whole-cell recordings from young flies demonstrated that phototransduction was unaffected, quantum bumps, macroscopic responses in the presence and absence of external Ca(2+), light adaptation, and Ca(2+) release from internal stores all being normal. Using the specific TRP channel blocker La(3+) we demonstrated that both TRP and TRPL channel functions were unaffected. These results indicate that InsP(3)R-mediated store depletion does not underlie TRP and TRPL activation in Drosophila photoreceptors.

Behavioural effects of receptor-specific substance P agonists

&NA; Septide and senktide are synthetic substance P (SP) agonists with extremely high selectivity for 1 of the 3 known SP receptor subtypes. When injected intrathecally, they produced dramatically different behavioural effects. Septide, the selective SP‐P receptor agonist, evoked intense, compulsive scratching, biting and licking of the hind limb, with no sign of motor flaccidity, and without measurable effect on responses to noxious thermal or mechanical stimulation of the foot or tail. In contrast, senktide, the selective SP‐N receptor agonist, produced profound, but transient, motor flaccidity, reduced response to noxious stimuli and, at low doses, ‘wet‐dog shakes.’ These various symptoms, all previously associated with SP and/or synthetic SP analogues, appear therefore to derive from activation of distinct SP receptor subtypes.

Phospholipase C and termination of G-protein-mediated signalling in vivo

In Drosophila photoreceptors, phospholipase C (PLC) and other signalling components form multiprotein structures through the PDZ scaffold protein INAD. Association between PLC and INAD is important for termination of responses to light; the underlying mechanism is, however, unclear. Here we report that the maintenance of large amounts of PLC in the signalling membranes by association with INAD facilitates response termination, and show that PLC functions as a GTPase-activating protein (GAP). The inactivation of the G protein by its target, the PLC, is crucial for reliable production of single-photon responses and for the high temporal and intensity resolution of the response to light.

Activation of Turkey erythrocyte adenylate cyclase and blocking of the catecholamine-stimulated GTPase by guanosine 5′-(γ-thio) triphosphate

Summary Experiments with GTP analogues tested how the catecholamine-stimulated GTPase relates to the adenylate cyclase of the turkey erythrocyte membrane. Incubation of the membranes with guanosine 5′-(γ-thio) triphosphate (GTPγS) and isoproterenol followed by washing resulted in persistent activation of the adenylate cyclase and in an inhibition of the catecholamine stimulated GTPase. Incubation of the membranes with GTPγS in the presence of propranolol did not cause activation of the adenylate cyclase nor did it inhibit the catecholamine-stimulated GTPase. The adenylate cyclase activated by guanosine 5′-(β, γ-imino) triphosphate (Gpp(NH)p), rapidly reverted (t½ = 1 min) to the basal inactive state upon incubation in a GTPase reaction mixture containing isoproterenol. Accordingly, preincubation with Gpp(NH)p and isoproterenol did not cause an inhibition of the catecholamine-stimulated GTPase. These findings suggest that both the catecholamine-stimulated GTPase and the binding of the guanyl nucleotides which activate the adenylate cyclase take place at the same regulatory site.

ATP-dependent calcium uptake by microsomal preparations from rat parotid and submaxillary glands.

Abstract Microsomal fractions isolated from rat parotid and submaxillary glands readily demonstrate ATP-dependent calcium uptake, Concentrations of inhibitors which completely block calcium uptake in mitochondria do not appreciably inhibit calcium uptake in the gland microsomes. Ouabain at 0.1 mM concentration does not inhibit calcium uptake in gland microsomes. The reaction shows a high degree of specificity for ATP as the nucleoside triphosphate which has a K m of 20 μM. The rate of calcium uptake per mg protein in the microsomal fraction is 25–30 nmoles per min at 30° and calcium storing capacity is 2.75 μmoles per mg protein. The indications that the microsomal vesicles are derived from the Golgi complex and the possible functions of calcium in this structure are discussed.

Regulation of light‐dependent Gqα translocation and morphological changes in fly photoreceptors

Heterotrimeric G‐proteins relay signals between membrane‐bound receptors and downstream effectors. Little is known, however, about the regulation of Gα subunit localization within the natural endogenous environment of a specialized signaling cell. Here we show, using live Drosophila flies, that light causes massive and reversible translocation of the visual Gqα to the cytosol, associated with marked architectural changes in the signaling compartment. Molecular genetic dissection together with detailed kinetic analysis enabled us to characterize the translocation cycle and to unravel how signaling molecules that interact with Gqα affect these processes. Epistatic analysis showed that Gqα is necessary but not sufficient to bring about the morphological changes in the signaling organelle. Furthermore, mutant analysis indicated that Gqβ is essential for targeting of Gqα to the membrane and suggested that Gqβ is also needed for efficient activation of Gqα by rhodopsin. Our results support the ‘two‐signal model’ hypothesis for membrane targeting in a living organism and characterize the regulation of both the activity‐dependent Gq localization and the cellular architectural changes in Drosophila photoreceptors.

The roles of trp and calcium in regulating photoreceptor function in Drosophila

Invertebrate photoreceptors use the ubiquitous inositol-lipid signaling pathway for phototransduction. This pathway depends on Ca2+ release from internal stores and on Ca2+ entry via light-activated channels to replenish the loss of Ca2+ in those stores. The Drosophila transient receptor potential (TRP) protein is essential for the high Ca2+ permeability and other biophysical properties of these light-activated channels, which affect both excitation and adaptation in photoreceptor cells. Physiological and heterologous expression studies indicate that TRP is a putative subunit of a surface membrane channel that can be activated by depletion of internal Ca2+ stores. Furthermore, trp is an archetypal member of a multigene family whose products share a structure that is highly conserved throughout evolution, from worms to humans.

Conversion of protein kinase to a cyclic AMP independent form by affinity chromatography on N6-caproyl 3′,5′-cyclic adenosine monophosphate-sepharose

Abstract A method was developed to couple 3′,5′-cyclic adenosine monophosphate (c-AMP) to Sepharose. Chromatography of protein kinases from rat parotid, and rabbit skeletal muscle on c-AMP-Sepharose, resulted in preparations which were fully active in the absence of c-AMP. The c-AMP independent kinases thus obtained no longer bind c-AMP. It is suggested that c-AMP-Sepharose activates the protein kinase by removal of a regulatory unit that binds c-AMP. So far no method has been found to recover the c-AMP binding unit from the c-AMP-Sepharose column.