Nullifying drug-induced sensitization: behavioral and electrophysiological evaluations of dopaminergic and serotonergic ligands in methamphetamine-sensitized rats.

Repeated exposure to methamphetamine produces a persistent enhancement of the acute motor effects of the drug, commonly referred to as behavioral sensitization. Behavioral sensitization involves monoaminergic projections to several forebrain nuclei. We recently revealed that the ventral pallidum (VP) may also be involved. In this study, we sought to establish if treatments with antagonists or partial agonists to monoaminergic receptors could "reverse" methamphetamine-induced behavioral and VP neuronal sensitization. Behavioral sensitization was obtained in rats with five once-daily s.c. injections of 2.5mg/kg methamphetamine, an effect that persisted for at least 60 days. After the development of sensitization, 15 once-daily treatments of mirtazapine (a 5-HT(2/3), alpha(2) and H(1) antagonist), SKF38393 (D(1) partial agonist) or SCH23390 (dopamine D(1) antagonist) nullified indices of motor sensitization as assessed by measuring the motoric response to an acute methamphetamine challenge 30 days after the fifth repeated methamphetamine treatment. VP neurons recorded in vivo from methamphetamine-sensitized rats at the 30-day withdrawal time also showed a robust downward shift in the excitatory responses observed to an acute i.v. methamphetamine challenge in non-sensitized rats. This decreased excitatory effect was reversed by mirtazapine, but not by other antagonists that were tested. These data suggest a potential therapeutic benefit for mirtazapine in the treatment of methamphetamine addiction, and point to a possible role for the VP in the sensitization process to methamphetamine.

PKC-dependent regulation of transepithelial resistance: roles of MLC and MLC kinase.

The mechanisms by which protein kinase C (PKC) activation results in increased transepithelial resistance (TER) are unknown [G. Hecht, B. Robinson, and A. Koutsouris. Am. J. Physiol. 266 (Gastrointest. Liver Physiol. 29): G214-G221, 1994]. We have previously shown that phosphorylation of the regulatory light chain of myosin II (MLC) is associated with decreases in TER and have suggested that contraction of the perijunctional actomyosin ring (PAMR) increases tight junction (TJ) permeability [J. R. Turner, B. K. Rill, S. L. Carlson, D. Carnes, R. Kerner, R. J. Mrsny, and J. L. Madara. Am. J. Physiol. 273 (Cell Physiol. 42): C1378-C1385, 1997]. We therefore hypothesized that PKC activation alters TER via relaxation of the PAMR. Activation of PKC by the phorbol ester phorbol 12-myristate 13-acetate (PMA) resulted in a progressive dose-dependent increase in TER that was apparent within 15 min (111% of controls) and maximal within 2 h (142% of controls). Similar increases were induced by a diacylglycerol analog, and the effects of both PMA and the diacylglycerol analog were prevented by the PKC inhibitor bisindolylmaleimide I. PMA treatment caused progressive decreases in MLC phosphorylation, by 12% at 15 min and 41% at 2 h. Phosphorylation of MLC kinase (MLCK) increased by 64% within 15 min of PMA treatment and was stable over 2 h (51% greater than that of controls). Thus increases in MLCK phosphorylation preceded decreases in MLC phosphorylation. These data suggest that PKC regulates TER via decreased phosphorylation of MLC, possibly due to inhibitory phosphorylation of MLCK. The decreased phosphorylation of MLC likely reduces PAMR tension, leading to decreased TJ permeability.

Transepithelial resistance can be regulated by the intestinal brush-border Na(+)/H(+) exchanger NHE3.

Initiation of intestinal Na(+)-glucose cotransport results in transient cell swelling and sustained increases in tight junction permeability. Since Na(+)/H(+) exchange has been implicated in volume regulation after physiological cell swelling, we hypothesized that Na(+)/H(+) exchange might also be required for Na(+)-glucose cotransport-dependent tight junction regulation. In Caco-2 monolayers with active Na(+)-glucose cotransport, inhibition of Na(+)/H(+) exchange with 200 microM 5-(N,N-dimethyl)- amiloride induced 36 +/- 2% increases in transepithelial resistance (TER). Evaluation using multiple Na(+)/H(+) exchange inhibitors showed that inhibition of the Na(+)/H(+) exchanger 3 (NHE3) isoform was most closely related to TER increases. TER increases due to NHE3 inhibition were related to cytoplasmic acidification because cytoplasmic alkalinization with 5 mM NH(4)Cl prevented both cytoplasmic acidification and TER increases. However, NHE3 inhibition did not affect TER when Na(+)-glucose cotransport was inhibited. Myosin II regulatory light chain (MLC) phosphorylation decreased up to 43 +/- 5% after inhibition of Na(+)/H(+) exchange, similar to previous studies that associate decreased MLC phosphorylation with increased TER after inhibition of Na(+)-glucose cotransport. However, NHE3 inhibitors did not diminish Na(+)-glucose cotransport. These data demonstrate that inhibition of NHE3 results in decreased MLC phosphorylation and increased TER and suggest that NHE3 may participate in the signaling pathway of Na(+)-glucose cotransport-dependent tight junction regulation.