Immunohistochemical study of opioid receptors after chronic morphine treatment in rats.

Previously, we have used the biochemical receptor binding method to investigate whether down-regulation of the opioid receptor is a mechanism for morphine tolerance, and we were led to a negative conclusion. In the current study, we further used immunohistochemistry to reinvestigate this issue. Male Sprague-Dawley rats (250-300 g) were chronically treated with morphine s.c. for 2, 4 or 6 days, using an escalating dosage paradigm (5-45 mg), which resulted in a 1.8 to 4.0-fold increase in AD50. Rat brains were removed, frozen, coronally sectioned (14 microm) and processed for mu- or delta-opioid receptor immunohistochemistry using the Avidin-Biotin Complex (ABC) method. No significant decrease in mu-opioid receptor (MOR) immunodensity was found in most of the brain regions, which were enriched with MOR after chronic treatment with morphine except for the anteroventral thalamic nucleus in the ventrolateral part (AVVL). No significant change in delta-opioid receptor (DOR) immunodensity after chronic treatment with morphine was found either. Therefore, our conclusion is that down regulation of opioid receptors may not be an important mechanism for morphine tolerance.

The role of nitric oxide in the development of morphine tolerance in rat hippocampal slices.

In the present study, we investigated the effects of a nitric oxide (NO) precursor, L-arginine, on the effect of different drugs, [trans-3, 4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamid e hydrochloride] (U-50,488, a kappa-opioid receptor agonist); dPTyr(Me)AVP (a vasopressin receptor antagonist); dizocilpine (MK-801, a N-methyl-D-aspartate (NMDA) receptor antagonist), to block the development of morphine tolerance or NO release in Sprague-Dawley rat hippocampal slices (450 microm). Slices were continuously superfused with artificial cerebrospinal fluid (ACSF) or drugs at 1 ml/min. Nichrome wire electrodes were placed in the Schaffer-collateral pathway and used to deliver biphasic 0.2-ms pulses of 5-30 V (0.033 Hz). A glass microelectrode was placed in the CA1 area to record population spikes. The amount of NO released in the superfusate was measured as nitrite formation. When the slices were superfused with 10 microM morphine, the amplitude of population spikes increased 200%-300% in 30-40 min. However, this effect of morphine decreased, i.e., tolerance developed, after continuous superfusion of morphine for 2-6 h. On the other hand, the nitrite level was increased about 250% of the control level through 6 h of morphine superfusion. Co-superfusion of L-arginine with morphine could further increase the nitrite level and also facilitate the development of morphine tolerance. On the other hand, 3-Br-7-nitroindazole (a neuronal NO synthase inhibitor) decreased the nitrite level significantly and blocked the development of morphine tolerance. When either U-50,488 (200 nM) or dPTyr(Me)AVP (500 pM) or MK-801 (500 pM) was co-superfused with morphine (10 microM), the development of morphine tolerance was blocked significantly and the nitrite level decreased to 100%-150% of the control level. L-arginine (500 nM) significantly reversed the effect of these drugs to block the development of morphine tolerance or to decrease the nitrite level through 6 h of superfusion. These data suggest that NO may play a key role in the development of morphine tolerance. Drugs which suppress the synthesis or release of NO would be expected to block the development of morphine tolerance.

Immunohistochemical evidence of down-regulation of mu-opioid receptor after chronic PL-017 in rats.

In a previous study, mu-opioid receptor binding was decreased by chronic treatment of rats with a mu-opioid receptor-selective agonist [CH3Phe3, D-Pro4]morphiceptin (PL-017) [Tao, P.L., Lee, H.Y., Chang, L.R., Loh, H.H., 1990. Decrease in mu-opioid receptor binding capacity in rat brain after chronic PL-017 treatment. Brain Res. 526, 270-275]. However, there was a lack of correlation between the time course of receptor down-regulation and the loss of pharmacological effects of the drug. In the current study, we used immunohistochemistry to reinvestigate this issue. Male Sprague-Dawley rats were chronically treated with PL-017 i.c.v. for 1, 3 or 5 days, using an escalating dosage paradigm (0.75-6.0 microg), which resulted in a 1.4 to 32-fold increase in the AD50. Rat brains were removed, frozen, coronally sectioned (14 microm) and processed for mu-, delta- or kappa-opioid receptor immunohistochemistry by the avidin-biotin complex (ABC) method. Significant decreases in OP3 immunodensity were found in many brain regions which are enriched with OP3 after chronic treatment of PL-017. Time-dependent decreases in OP3 were detected and reached a plateau around 3 days of PL-017 treatment. No significant change in OP1 or OP2 immunodensity after chronic treatment with PL-017 was found. Our conclusion is that chronic treatment with PL-017 of rats selectively down-regulates mu-opioid receptors in the brain. This may be an important mechanism for PL-017 tolerance.

Blockade of the development of morphine tolerance by U-50,488, an AVP antagonist or MK-801 in the rat hippocampal slice.

1. In this study, we investigated the effects of different drugs (a kappa-opioid receptor agonist U-50,488, a vasopressin receptor antagonist dPTyr(Me)AVP or an N-methyl-D-aspartate (NMDA) receptor antagonist MK-801) on the development of morphine tolerance in rat hippocampal slices. 2. Hippocampal slices (450 microm) of Sprague-Dawley rats (250-300 g) were used. Slices were continuously superfused with artificial CSF or drugs at 1 ml min(-1). Nichrome wire electrodes were placed in the Schaffer-collateral pathway and used to deliver biphasic 0.2 ms pulses of 5-30 V (0.033 Hz). A glass microelectrode was placed in the CA1 area to record population spikes. 3. When the slices were superfused with 10 microM morphine, the amplitude of population spikes increased 2-3 fold in 30-40 min. However, this effect of morphine decreased, i.e. tolerance developed after continuous superfusion of morphine for 2-6 h. 4. When either U-50,488 (200 nM) or dPTyr(Me) AVP (500 pM) or MK-801 (500 pM) was co-superfused with morphine (10 microM), it significantly blocked the development of morphine tolerance. Nor-BNI (a kappa-opioid receptor antagonist, 200 nM) significantly reversed the inhibitory effect of U-50,488 but not those of dPTyr(Me)AVP or MK-801 on the development of morphine tolerance. 5. These data indicate that kappa-opioid receptors, AVP receptors and NMDA receptors are all involved in the development of morphine tolerance. The suppression of kappa-opioid receptor activity after chronic morphine may occur before the activation of AVP receptors or NMDA receptors during the development of morphine tolerance.

Alteration of the interaction of mu-opioid receptor and G protein after chronic PL017 treatment in rats.

Alteration in ligand-receptor interaction during chronic drug treatment has been suggested as a possible mechanism underlying opioid tolerance. However, our previous studies found that chronic PL017 (a selective mu-opioid agonist) treatment of adult animals resulted in down regulation of mu opioid receptor levels only after 5 days of PL017 treatment although tolerance had significantly developed after 3 days of PL017 treatment. Since G protein seems to be involved in regulation of opioid receptors, we suspect that opioid receptor-G protein interaction may be altered after chronic PL017 treatment before down-regulation of opioid receptors occurrs. Our investigation proceeded first, by measuring the ability of Gpp(NH)p to alter mu-opioid agonist: [3H]DAMGO binding; and second, by measuring the opioid agonist-stimulated GTPase activity before and after chronic PL017 treatment for 1 or 3 days when tolerance has developed but without down-regulation. We found that after 1 day and 3 days of PL017 treatment, rats produced 1.9 and 7.4 fold degree of tolerance. In receptor binding assay, we found the Bmax values did not show significant difference before and after chronic PL017 treatment. On the other hand, 10 microM Gpp(NH)p (a stable GTP analogue) significantly increased the Kd of the control midbrain by 2.59 +/- 0.21 fold but only increased the Kd by 1.92 +/- 0.11 fold after 3 days of PL017 treatment. Furthermore, the EC50 and maximal effect of DAMGO on stimulating low Km GTPase activity for control midbrain are 1.2 +/- 0.3 10(-8) M and 21.7 +/- 0.6%, respectively; in the experimental group, after 3 days PL017 treatment, the EC50 has increased to 7.3 +/- 2.7 x 10(-8) M and maximal stimulation decreased to 16.6 +/- 1.1%. The present findings indicate that after 3 days chronic PL017 treatment: (1) The effect of Gpp(NH)p on the affinity of mu-opioid receptor and DAMGO has been diminished. (2) The effect of DAMGO on stimulating low Km GTPase activity of G protein has been decreased. Therefore, it seems that the interaction between opioid receptor and G protein has been altered after chronic PL017 treatment. This phenomenum happens before down-regulation, and it may be one of the mechanisms for opioid tolerance.

Abnormal electrical properties of myocytes from chronically infarcted canine heart. Alterations in Vmax and the transient outward current.

BACKGROUND: Reentrant ventricular arrhythmias can occur in the surviving muscle fibers of the epicardial border zone of the canine heart 5 days after coronary artery occlusion. To understand the cellular basis of these arrhythmias, we developed a method of dispersing myocytes (IZs) from the epicardial border zone. METHODS AND RESULTS: We compared the electrophysiological properties of IZs with those of cells dispersed from the epicardium of control noninfarcted (NZs) and of sham-operated animals (NZsham). Transmembrane action potentials of IZs are reduced in total action potential amplitude and maximum upstroke velocity compared with NZs. However, resting potential of IZs is no different from that of NZs. Action potential duration at -10 mV is significantly reduced in IZs compared with control, and IZ potentials do not show the typical "spike and dome" morphology that is evident in all NZs. Using Vmax as an indirect measure of the peak inward current available for the upstroke of the action potential, we found that the availability curve for IZs is significantly different from the NZ curve. Furthermore, the time course of recovery of Vmax after a depolarizing voltage clamp step was significantly altered in IZs. Using whole-cell voltage clamp techniques, we determined that the voltage-dependent, Ca(2+)-independent, 4-aminopyridine-sensitive transient outward current (ito1) occurred in all NZs (n = 16) but existed in only 37% of IZs (n = 16). There was a significant reduction in the density of ito1 elicited by depolarizing steps in those IZs showing ito1 compared with ito1 density in NZs. CONCLUSIONS: We have developed a single-cell model of cells that survive in the infarcted heart. Our studies indicate that there are changes in Vmax in IZs. In addition, there is no prominent phase 1 of repolarization in IZ action potentials. This is consistent with the dramatic loss in the function of the ionic channel responsible for the voltage-dependent transient outward current, ito1.