Protein phosphorylation, mediated by protein kinases, plays a crucial role in cellular regulation. One of the most important protein kinases is protein kinase A (PKA). N-[2-p-bromocinnamylamino-ethyl]-5-isoquinolinesulphonamide (H89) is often used as a “PKA specific inhibitor” to study the involvement of PKA in signaling pathways. However, evidence from cell-free experiments has suggested that H89 can also inhibit other protein kinases. In this study, previously generated PKA-null and PKA-intact mouse cell lines derived from mpkCCD cells were treated with H89 over a range of concentrations commonly used in the literature, followed by mass spectrometry-based phosphoproteomics to globally assess changes in phosphorylation. From a total of 14,139 phosphorylation sites quantified, we found that 571 and 263 phosphorylation sites with significant changes in abundance in PKA-intact and PKA-null cells, respectively. Analyses of sequence logos generated from significantly decreased phosphorylation sites in PKA-intact and PKA-null cells both revealed a preference for basic amino acids at position −3 and −2. Thus, H89 appears to inhibit basophilic kinases even in the absence of PKA. Likely H89 targets include basophilic protein kinases such as AKT, RSK, AMPK and ROCK. We conclude that, in intact cells, H89 can affect activities of protein kinases other than PKA, and therefore responses to H89 should not be regarded as sufficient evidence for PKA involvement in a signaling process.

Sex differences in the prevalence of dopamine-related neuropsychiatric diseases and in the sensitivity to dopamine-boosting drugs such as stimulants is well recognized. Here we assessed whether there are sex differences in the brain dopamine system in humans that could contribute to these effects. We analyzed data from two independent [11C]raclopride PET brain imaging studies that measured methylphenidate-induced dopamine increases in the striatum using different routes of administration (Cohort A = oral 60 mg; Cohort B = intravenous 0.5 mg/kg; total n = 95; 65 male, 30 female), in blinded placebo-controlled designs. Females when compared to males reported stronger feeling of “drug effects” and showed significantly greater dopamine release in the ventral striatum (where nucleus accumbens is located) to both oral and intravenous methylphenidate. In contrast, there were no significant differences in methylphenidate-induced increases in dorsal striatum for either oral or intravenous administration nor were there differences in levels of methylphenidate in plasma. The greater dopamine increases with methylphenidate in ventral but not dorsal striatum in females compared to males suggests an enhanced sensitivity specific to the dopamine reward system that might underlie sex differences in the vulnerability to substance use disorders and to attention-deficit/hyperactivity disorder (ADHD).

The continuous rise in opioid overdoses in the United States is predominantly driven by very potent synthetic opioids, mostly fentanyl and its derivatives (fentanyls). Although naloxone (NLX) has been shown to effectively reverse overdoses by conventional opioids, there may be a need for higher or repeated doses of NLX to revert overdoses from highly potent fentanyls. Here, we used positron emission tomography (PET) to assess NLX’s dose-dependence on both its rate of displacement of [11C]carfentanil ([11C]CFN) binding and its duration of mu opioid receptor (MOR) occupancy in the male rat brain. We showed that clinically relevant doses of intravenously (IV) administered NLX (0.035 mg/kg, Human Equivalent Dose (HED) 0.4 mg; 0.17 mg/kg, HED 2 mg) rapidly displaced the specific binding of [11C]CFN in the thalamus in a dose-dependent manner. Brain MOR occupancy by IV NLX was greater than 90% at 5 min after NLX administration for both doses, but at 27.3 min after 0.035 mg/kg dose and at 85 min after 0.17 mg/kg NLX, only 50% occupancy remained. This indicates that the duration of NLX occupancy at MORs is short-lived. Overall, these results show that clinically relevant doses of IV NLX can promptly displace fentanyls at brain MORs, but repeated or higher NLX doses may be required to prevent re-narcotization following overdoses with long-acting fentanyls.