Changes in free cytosolic calcium and accumulation of inositol phosphates in isolated hepatocytes by [Leu]enkephalin

Abstract

Isolated hepatocytes from fed rats were used to study the effects of the opioid peptide [Leu]enkephalin on intracellular free cytosolic Ca2+ ([Ca2+]i) and inositol phosphate production. By measuring the fluorescence of the intracellular Ca2+-selective indicator quin-2, [Leu]enkephalin was found to increase [Ca2+]i rapidly from a resting value of 0.219 microM to 0.55 microM. The magnitude of this response was comparable with that produced by maximally stimulating concentrations of either vasopressin (100 nM) or phenylephrine (10 microM). The opioid-peptide-mediated increase in [Ca2+]i showed a dose-dependency comparable with the activation of phosphorylase, but it preceded the increase in phosphorylase alpha activity. Addition of [Leu]enkephalin to hepatocytes prelabelled with myo-[2-3H(n)]inositol resulted in a significant stimulation of inositol phosphate production. At 10 min after hormone addition, there were increases in the concentrations of inositol mono-, bis- and tris-phosphate fractions of 12-, 9- and 14-fold respectively. No effect was apparent on the glycerophosphoinositol fraction. The effect of 10 microM-[Leu]enkephalin on inositol phosphate production was significantly greater than that obtained with 10 microM-phenylephrine, but marginally smaller than that induced by 100 nM-vasopressin. However, at these concentrations all three agonists gave a comparable increase in [Ca2+]i and activation of phosphorylase a. These data provide evidence for [Leu]enkephalin acting via a mechanism involving a mobilization of Ca2+ as a result of increased phosphatidylinositol turnover.

δ‐Opioids Stimulate Inositol 1,4,5‐Trisphosphate Formation, and so Mobilize Ca2+ from Intracellular Stores, in Undifferentiated NG108‐15 Cells

“…These data clearly indicate that in undifferentiated NGIO8-15 cells, b-opioids activate PLC via a pertussis toxin-sensitive 0 protein and so stimulate Ins (1.4,5) P 3 formation, which mobilizes intracellular Ca 25. Similar findings have been reported for K-opioids in chick and rat cardiomyocytes (Ventura et a!., 1992;Wong et al, 1995) and O-opioids in rat hepatoeytes (Leach et al, 1986). It is of interest that the current data show the coupling of the 6-opioid receptor to PLC via G~,,, but we are unable to comment whether this is a direct coupling or mediated by~y subunits.…”

“…Nevertheless. opioids have been shown to activate PLC, and thus generate lns(l,4.5)P2, in other cell types (Leach et a!.. 1986;Pcriyasamy and I-loss, 1990;Johnson et a!., l994;Smart et al, 1994), including cells transfected with the cloned 6-opioid receptor (Miyairtae et al, 1993;Tsu eta!., 1995).…”

The occurrence and receptor specificity of endogenous opioid peptides within the pancreas and liver of the rat. Comparison with brain

“…A centrally mediated sympathetic connection between opioid peptides and glucose homoeostasis has been established for some time (Bodo et al, 1937;Vassalle, 1961;Feldberg & Smyth, 1977;Pfeiffer et al, 1983); however, evidence for a direct peripheral action of opiates in the regulation of hepatic glucose production and on glucose homoeostasis through pancreaticislet hormone secretion has only more recently been reported (Ipp et al, 1978;Green et al, 1980Green et al, , 1983aAllan et al, 1983;Leach et al, 1985; Leach & Titheradge, 1986;Hothi et al, 1988). /J-Endorphin, [Met5]enkephalin, [Leu5]enkephalin and dynorphin A-(1-13)-peptide have been shown to regulate insulin, glucagon and somatostatin release from isolated islets of Langerhans by a naloxone-reversible mechanism (Ipp et al, 1978;Green et al, 1980Green et al, , 1983aHermansen, 1983;Curry et al, 1987;Toyota et al, 1985).…”

“…/8-Endorphin (Matsumura et al, 1984), [Met5]enkephalin, [Leu5]enkephalin and dynorphin A-(l-l 3)-peptide (Allan et al, 1983;Leach et al, 1985;Leach & Titheradge, 1986) have all been shown to induce a rapid dose-dependent stimulation of both glycogenolysis and glycogenesis. The mechanism of action of ,-endorphin has been attributed to a rise in cyclic AMP (Matsumura et al, 1984), whereas the enkephalins and dynorphin decrease cyclic AMP levels and adenylate cyclase activity and increase inositol lipid turnover and cytosolic Ca2l (Leach & Titheradge, 1986;Leach et al, 1986;Hothi et al, 1988). Wajda et al (1976) have identified the existence of an opiate-like factor in Tris/HCl extracts of rat liver by its ability to displace [3H]naloxone and [3H]dihydromorphine from rat brain membrane receptors in a dose-dependent fashion.…”

Attenuation of morphine tolerance after antisense oligonucleotide knock‐down of spinal mGluR1

“…A role of mGluRs in opioid tolerance is not unexpected, since previous studies have demonstrated that opioids influence both glutamate transmission and glutamate‐linked second messengers, and vice‐versa (Fundytus & Coderre, 1999a,b). Thus, opioids have been found to activate PLC (Okajima et al ., 1993; Smart et al ., 1995; Tsu et al ., 1995), stimulate PI hydrolysis (Leach et al ., 1986; Periyasamy & Hoss, 1990; Smart et al ., 1994, and increase intracellular Ca 2+ release (Jin et al ., 1992), as well as increasing PKC (Kramer & Simon, 1999; Narita et al ., 1994b). Furthermore, PKC stimulates the secretion of β‐endorphin in pituitary and hypothelamic neurons (Abou‐Samira et al ., 1987; Kapcala et al ., 1992).…”