Two isoforms of the substance P (SP) receptor, differing in the length of the cytoplasmic carboxylterminus by Ϸ8 kDa, have been detected previously in rat salivary glands and other tissues. The binding and functional properties of these two isoforms have been investigated using full-length (407 amino acids) and carboxyl-terminally truncated ( These differences in responsiveness may be related to the observed differences in receptor desensitization. The truncated receptor, in contrast to the full-length receptor, does not undergo rapid and long-lasting desensitization. Cells possessing the short isoform of the SP receptor would thus be expected to exhibit a prolonged responsiveness.Substance P (SP), a member of the tachykinin family of peptides, is involved in many physiological processes, including exocrine gland secretion, vasodilation, pain transmission, and neurogenic inflammation (1). Molecular cloning established that the receptor for SP belongs to the G protein-coupled receptor family that possesses seven putative transmembrane domains (2, 3). Results from crosslinking and reconstitution studies have provided evidence that the SP receptor, also known as the neurokinin-1 receptor, couples to a subgroup of G proteins, Gq͞ 11 (4, 5). It has been shown in many tissues that SP activates phospholipase C ␤ (PLC ␤ ) which results in a transient increase in intracellular inositol 1,4,5-trisphosphate (IP 3 ) and cytosolic calcium concentration (6-9). The SP receptor, when stably expressed in Chinese hamster ovary (CHO) cells, also couples to phosphatidylinositol 4,5-bisphosphate (PIP 2 ) hydrolysis (10, 11).Biochemical studies in which the SP receptors in a membrane preparation of rat submaxillary glands were covalently labeled with a photoreactive SP analog (12, 13) have demonstrated the presence of two SP receptor isoforms that differ in the length of the cytoplasmic carboxyl-terminus by approximately 8 kDa. The photolabeling of the full-length SP receptor isoform (molecular mass, 46 kDa following deglycosylation) and the short isoform (molecular mass, 37 kDa following deglycosylation) are inhibited by SP with the same IC 50 (1 nM), indicating that both receptor isoforms bind SP with a similar affinity. A cDNA encoding a carboxyl-terminally truncated SP receptor with a calculated molecular weight of 35,797 has been cloned (14). When expressed in COS cells, this truncated receptor, in contrast to the receptor detected by photoaffinity labeling, has a binding affinity at least 10-fold less than that of the full-length receptor. Therefore, it is unlikely that the short receptor isoform identified in the rat salivary gland is derived from this spliced variant.To study the functional properties of the short SP receptor isoform identified in the rat salivary gland by photoaffinity labeling, a rat SP receptor mutant (324 amino acids; molecular weight, 37,360) carboxyl-terminally truncated to approximate the size of this short receptor isoform (13) was stably expressed in CHO cells (15). In this report, we have co...

Propranolol, a cationic amphiphilic drug, caused enhanced incorporation of labeled precursor into phosphatidic acid and its metabolites in rat cerebral cortex mince, suggesting increased biosynthesis or reduced degradation. Inhibition of phosphatidate phosphohydrolase could explain the observed drug-induced accumulation of phosphatidic acid and other acidic lipids. Propranolol exhibited differential effects on the free and membrane-bound forms of phosphatidate phosphohydrolase. The drug inhibited cytoplasmic enzyme in a dose-dependent manner only when membrane-bound substrate was used but had practically no effect on the membrane-bound enzyme irrespective of the nature of the substrate used or on the cytoplasmic enzyme when free substrate was used. Brain cytoplasmic enzyme obtained from rats sacrificed 30 min after intraperitoneal injections of propranolol did not show any inhibition. propranolol bound to membranes may prevent cytoplasmic enzyme action, probably by decreasing the availability of substrate through the formation of stable lipid-drug-protein complexes.

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Certain biological actions of phorbol esters cannot be duplicated by diacylglycerol (DAG). Thus, the human neuroblastoma cell line SH-SY5Y differentiates when exposed to 12-tetradecanoyl-13-acetyl-beta-phorbol (TPA) and protein kinase C (PKC) inhibitors, but not when exposed to DAG. To investigate the specific features of the phorbol diester molecule that might be responsible for these effects, we examined the extension of neurites, expression of neuron-specific enolase, and appearance and localization of phosphorylated high molecular weight neurofilament subunits (NF-H). TPA, 12-deoxy-13-tetradecanoyl-beta-phorbol, and staurosporine, but not DAG or 4-O-methyl-TPA, caused neurite outgrowth. Neuron-specific enolase was expressed in cells treated with TPA and 12-deoxy-13-tetradecanoyl-beta-phorbol but not with DAG, staurosporine, or 4-O-methyl-TPA. NF-H increased in the perikarya of cells treated with DAG and 4-O-methyl-TPA, in processes and to varying degrees in perikarya of TPA- and 12-deoxy-13-tetradecanoyl-beta-phorbol-treated cells, but much more in the processes than in the perikarya of staurosporine-differentiated cells. These findings and additional differences between the differentiation induced by TPA (a PKC activator) and staurosporine (a PKC inhibitor), including distinct morphology of the cell body and processes and time of appearance of the morphological phenotype, suggest that activators and inhibitors of PKC induce differentiation of SH-SY5Y cells by different mechanisms, and that the five-membered/seven-membered terpene ring region present in TPA must be intact for the induction of morphological differentiation.

A decrease in protein kinase C activity caused either by treatment with inhibitors, such as staurosporine or H-7, or by prolonged exposure to phorbol diesters has been proposed to be involved in the early events of SH-SY5Y neuroblastoma cell differentiation. Because eight distinct isoforms of protein kinase C with discrete subcellular and tissue distributions have been described, we determined which isoforms are present in SH-SY5Y cells and studied their modifications during differentiation. The alpha, beta 1, delta, and epsilon isoforms were present in SH-SY5Y cells, as well as in rat brain. Protein kinase C-alpha and -beta 1 were the most abundant isoforms in SH-SY5Y cells, and immunoreactive protein kinase C-delta and -epsilon were present in much smaller amounts than in rat brain. Subcellular fractionation and immunocytochemistry demonstrated that all four isoforms are distributed bimodally in the cytoplasm and the membranes. Immunocytochemical analysis showed that the alpha isoform is associated predominantly with the plasma membrane and the processes extended during treatment with 12-tetradecanoyl-13-acetyl-beta-phorbol or staurosporine, and that protein kinase C-epsilon is predominantly membrane-bound. Its localization did not change during differentiation. Western blots of total SH-SY5Y cell extracts and of subcellular fractions probed with isoform-specific polyclonal antibodies showed that when SH-SY5Y cells acquired a morphologically differentiated phenotype, protein kinase C-alpha and -epsilon decreased, and protein kinase C-beta 1 did not change. These data suggest distinct roles for the different protein kinase C isoforms during neuronal differentiation, as well as possible involvement of protein kinase alpha and epsilon in neuritogenesis.

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