Leftward Shift In Concentration-response Curve Research Articles

Pharmacological studies on the "orphan" opioid receptor in central and peripheral sites

We have exploited the availability of the "orphan" opioid receptor (referred to here as ORL1) in its "natural state" to investigate the effect of nociceptin (orphanin FQ), the endogenous agonist for the ORL1 receptor in the brain, vas deferens, and myenteric plexus of the small intestine. Nociceptin was a potent agonist in electrically stimulated preparations of vasa deferentia (rat and rabbit) and myenteric plexus (guinea-pig) (IC50 ranging from 18 to 31 nM) and susceptible to enzymic cleavage as addition of a cocktail of peptidase inhibitors to the organ bath produced a leftward shift in concentration-response curves (IC50 ranging from 2.1 to 4.9 nM). In radioligand binding experiments using brain membranes from rat, rabbit, and guinea-pig, [3H]nociceptin bound a single population of binding sites with high affinity (KD values ranging from 0.049 to 0.124 nM) and capacity (Bmax ranging from 143 to 254 fmol.mg-1 protein). However, the response to nociceptin in functional studies and in radioligand binding inhibitory assays was resistant to antagonism/displacement by naloxone and a range of other opioid receptor antagonists, thus displaying a very different pharmacological profile from that of the "classical" opioids. Therefore, we conclude that the effect of nociceptin in these studies is not via an action at mu, delta, or kappa opioid receptors but rather at an orphan opioid receptor, ORL1.

Pharmacological studies on the "orphan" opioid receptor in central and peripheral sites

We have exploited the availability of the "orphan" opioid receptor (referred to here as ORL1) in its "natural state" to investigate the effect of nociceptin (orphanin FQ), the endogenous agonist for the ORL1 receptor in the brain, vas deferens, and myenteric plexus of the small intestine. Nociceptin was a potent agonist in electrically stimulated preparations of vasa deferentia (rat and rabbit) and myenteric plexus (guinea-pig) (IC50 ranging from 18 to 31 nM) and susceptible to enzymic cleavage as addition of a cocktail of peptidase inhibitors to the organ bath produced a leftward shift in concentration-response curves (IC50 ranging from 2.1 to 4.9 nM). In radioligand binding experiments using brain membranes from rat, rabbit, and guinea-pig, [3H]nociceptin bound a single population of binding sites with high affinity (KD values ranging from 0.049 to 0.124 nM) and capacity (Bmax ranging from 143 to 254 fmol ·mg-1 protein). However, the response to nociceptin in functional studies and in radioligand binding inhibitory assays was resistant to antagonism/displacement by naloxone and a range of other opioid receptor antagonists, thus displaying a very different pharmacological profile from that of the "classical" opioids. Therefore, we conclude that the effect of nociceptin in these studies is not via an action at µ, delta , or kappa opioid receptors but rather at an orphan opioid receptor, ORL1.Key words: nociceptin, orphanin FQ, vas deferens, myenteric plexus, "orphan" opioid receptor.

Selective potentiation of histamine H 1-receptor stimulated calcium responses by 1,4-dithiothreitol in DDT 1MF-2 cells

The effect of 1,4-dithiothreitol (DTT) on agonist-stimulated increases in intracellular free calcium concentration ([Ca 2+] i) has been investigated in the smooth muscle cell line, DDT 1MF-2, derived from hamster vas deferens. Pretreatment with DTT (1 mM) produced a large leftward parallel shift in concentration-response curve for histamine H 1-receptor mediated increases in [Ca 2+] i. The ec 50 values for H 1-receptor stimulated increases in [Ca 2+] i in the absence and presence of DTT were 11.3 ± 1.5 μM (N = 6) and 0.52 ± 0.15 μM (N = 6), respectively. DTT had no significant effect on the maximum Ca 2+ response elicited by histamine (100 μM). In the presence of DTT the partial H 1-receptor agonist 2-pyridylethylamine (100 μM) increased [Ca 2+] i from 112 ± 14 nM to 237 ± 24 nM (N = 10). In control cells 2-pyridylethylamine (100 μM) did not elicit a Ca 2+ response. DTT had no significant effect on the maximum Ca 2+ response elicited by 1 mM 2-pyridylethylamine. The enhancement of histamine H 1-receptor Ca 2+ responses by DTT was reversed by the sulphydryl oxidizing agent dithiobis-(2-nitrobenzoic acid). DTT had no significant effect on adenosine A 1-, bradykinin and ATP-receptor stimulated increases in [Ca 2+] i. [ 3H]mepyramine binding experiments confirmed that DTT increased agonist affinity. DTT produced a small, but significant, leftward shift in concentration-response curve for histamine displacement of [ 3H]mepyramine binding. These data suggest that DTT potentiates H 1-receptor mediated Ca 2+ responses by increasing agonist affinity.

Extracellular Ca(2+)-dependent potentiation by cocaine of serotonin- and norepinephrine-induced contractions in rat vascular smooth muscle.

Using front-surface fluorometry, we determined the effects of cocaine on force and cytosolic Ca2+ concentration ([Ca2+]i) in the rat aorta. We also examined the effects of cocaine on 45Ca2+ influx. Cocaine (10(-7) to 10(-4) M) alone did not alter the resting level of [Ca2+]i and force. Cocaine (< 10(-4) M), in a concentration-dependent manner, potentiated the 10(-6) M serotonin (5-HT)-induced or 10(-8) M norepinephrine (NE)-induced sustained increase in [Ca2+]i and force in the presence of extracellular Ca2+, whereas it had no potentiating effects in Ca(2+)-free solution. Similar potentiating effects of cocaine were observed in pharmacologically denervated strips. Cocaine (10(-5) M) produced a leftward shift of concentration-response curves for both 5-HT- and NE-induced increases in [Ca2+]i and force with no effect on the maximal response or the relations between [Ca2+]i and force. Cocaine (10(-5) M also accelerated the 45Ca2+ influx during activation by 10(-6) M 5-HT or by 10(-8) M NE. Cocaine (> 10(-3) M) inhibited 5-HT-, NE-, and high-K+ depolarization-induced contractions accompanied by decreases in [Ca2+]i in normal physiological salt solution and 5-HT- or NE-induced transient increase in [Ca2+]i and force in Ca(2+)-free physiological salt solution. Thus, low concentrations of cocaine potentiate NE- or 5-HT-induced contraction by augmenting the increase in [Ca2+]i. These potentiating effects may derive from either an increase in the affinity of the receptors to agonists or an increase in the Ca2+ influx. On the other hand, high concentrations of cocaine (> 10(-3) M) have a relaxant effect on vascular smooth muscle, as a result of a decrease in [Ca2+]i.

Effects of α2-Adrenergic agonist preincubation on subsequent forskolin-stimulated adenylate cyclase activity and [ 3H]forskolin binding in membranes from HT29 cells

α 2-Adrenergic agonist preincubation resulted in a leftward shift in the subsequent concentration-response curve to forskolin-stimulated adenylate cyclase activity in membranes from HT29 cells, a human colon adenocarcinoma cell line. This effect was much less pronounced than the effect seen in the intact cell cyclic AMP production assays. Removal of GTP from the assay caused a further slight leftward shift in the concentration-response curve. In [ 3H]forskolin binding experiments, α 2-adrenergic agonist preincubation caused a doubling of the maximal number of binding sites (80 vs 31 fmol/mg protein) compared to control. The addition of MgCl 2 and NaF to the assay buffer increased control binding 5-fold. With agonist preincubation, there was a further increase in binding in the presence of MgCl 2 and NaF which was not significantly different from the appropriate control. Pertussis toxin pretreatment blocked both the leftward shift in the forskolin concentration-response curve and the increase in maximal number of binding sites, indicating that a pertussis toxin sensitive protein is involved in these changes. Activation of cyclic AMP production in the intact cell by cholera toxin followed by norepinephrine preincubation and then stimulation by forskolin resulted in a degree of sensitization similar to that seen in the membrane adenylate cyclase and binding assays. Pertussis toxin also blocked this sensitization. It appears that if the cyclase system is highly activated, then the degree of sensitization is similar in the membrane and intact cell assay.