Evidence for biphasic effects of protein kinase C on serotonin transporter function, endocytosis, and phosphorylation

The serotonin transporter (SERT) regulates 5-hydroxytryptamine (serotonin) (5-HT) neurotransmission and is a high-affinity target for antidepressants and psychostimulants. In the present study, we investigated the mechanisms that contribute to a previously unidentified biphasic regulation of endogenous SERTs expressed in the platelets. Treatment of rat platelets with beta-phorbol 12-myristate 13-acetate (PMA) for 5 min or less resulted in a rapid inhibition of SERT involving changes in intrinsic activity of the transporter (increased K(m) and decreased V(max)). beta-PMA treatment for 30 min or more produced a sustained inhibition of SERT with a decrease only in the V(max). Whereas inhibition of SERT activity was detected from 1 to 45 min after phorbol ester addition, the decrease in surface SERT required at least 30 min of phorbol ester incubation. Increased endocytosis of SERT accounted for the decrease in surface SERT at the later point. Protein kinase C (PKC)-mediated phosphorylation of SERT occurs on the plasma membrane during the initial phase of rapid transporter inhibition, and later, the phosphorylated SERT enters the intracellular pool. beta-PMA-induced phosphorylation of SERT occurs initially on serine residues(s) and then on threonine residue(s). The initial serine phosphorylation corresponded to the first phase of rapid inhibition mediated by changes in intrinsic activity and/or silencing of SERT. The later phosphorylation on threonine residue(s) corresponded to the later phase of sustained inhibition mediated by an enhanced endocytosis of SERT. Together, these data reveal that in platelets, SERT function is regulated by PKC in a biphasic manner involving both trafficking-dependent and independent mechanisms and that these two events occur at distinct phases of transporter phosphorylation.     

The smoking-mortality association varies over time and by ethnicity in New Zealand

BACKGROUND: The strength of the smoking-mortality association may vary over time and by ethnic group. METHODS: Cohort studies of 1.6 million (1981-84) and 1.9 million (1996-99) New Zealanders aged 25-74 years were formed by the linkage of census and mortality data. Comparing current smokers with never smokers, standardized rate ratios (RRs) and rate differences (RDs) were calculated for all-cause and ischaemic heart disease (IHD) mortality. RESULTS: Between 1981-84 and 1996-99 the all-cause mortality RR increased from 1.59 (95% CI 1.53-1.66) to 2.05 (1.97-2.14) for men and from 1.49 (1.42-1.56) to 2.01 (1.91-2.12) for women. All-cause RRs were significantly greater among non-Ma-ori non-Pacific than Ma-ori: 2.22 (2.12-2.33) compared with 1.51 (1.35-1.69) in men and 2.20 (2.09-2.33) compared with 1.45 in women (1.27-1.66), respectively, in 1996-99. This RR heterogeneity remained after adjusting for socio-economic factors and was similar for IHD. The RDs demonstrated less heterogeneity. For example, in 1996-99 the RDs were 627 per 100,000 (452-802) for Ma-ori compared with 464 (427-502) for non-Ma-ori non-Pacific among men, and 368 (228-509) compared with 340 (311-370) among women. CONCLUSIONS: In New Zealand the relative effect of smoking on mortality differs over time and by ethnicity. We expect that such heterogeneity exists in other countries where the background mortality rates vary over time or between social groups. Information on this heterogeneity, including ethnicity-specific data, is needed to accurately determine the mortality burden owing to tobacco. The size of the RR estimates should be interpreted in the context of absolute mortality and effect measures.     

Early-response biological dosimetry--recommended countermeasure enhancements for mass-casualty radiological incidents and terrorism

The effective medical management of a suspected acute radiation overexposure incident necessitates recording dynamic medical data, measuring appropriate radiation bioassays, and estimating dose from dosimeters and radioactivity assessments in order to provide diagnostic information to the treating physician and a dose assessment for personnel radiation protection records. The accepted generic multiparameter and early-response approach includes measuring radioactivity and monitoring the exposed individual; observing and recording prodromal signs/symptoms and erythema; obtaining complete blood counts with white blood cell differential; sampling blood for the chromosome-aberration cytogenetic bioassay using the "gold standard" dicentric assay (translocation assay for long times after exposure) for dose assessment; bioassay sampling, if appropriate, to determine radioactivity contamination; and using other available dosimetry approaches. In the event of a radiological mass-casualty incident, current national resources need to be enhanced to provide suitable dose assessment and medical triage and diagnoses. This capability should be broadly based and include stockpiling reagents and devices; establishing deployable (i.e., hematology and biodosimetry) laboratories and reference (i.e., cytogenetic biodosimetry, radiation bioassay) laboratories; networking qualified reference radioactivity-counting bioassay laboratories, cytogenetic biodosimetry, and deployable hematology laboratories with the medical responder community and national radiation protection program; and researching efforts to identify novel radiation biomarkers and develop applied biological dosimetry assays monitored with clinical, deployable, and hand-held analytical systems. These research and applied science efforts should ultimately contribute towards approved, regulated biodosimetry devices or diagnostic tests integrated into a national radioprotection program.     

Developmental expression of the high affinity choline transporter in cholinergic sympathetic neurons

Choline uptake by the high affinity choline transporter (CHT) is the rate-limiting step in acetylcholine synthesis. Induction of CHT is therefore a critical step in cholinergic differentiation, and we examined the developmental expression of CHT in cholinergic sympathetic neurons that innervate rodent sweat glands. During postnatal development the earliest sympathetic axons in the rear footpads are noradrenergic, containing intense tyrosine hydroxylase immunoreactivity and lacking CHT-immunoreactivity (CHT-IR). By postnatal day 7 (P7) in mouse, and P10 in rat, weak CHT-IR appeared in axons associated with the sweat gland anlagen. CHT staining intensity increased during the following weeks in conjunction with plexus arborization and gland maturation. The pattern of CHT-immunoreactivity (CHT-IR) in the sweat gland innervation was similar to staining for the vesicular acetylcholine transporter and vasoactive intestinal peptide. Immunoblots of tissue from sympathectomized rats confirmed that most of the CHT in footpad was contained in sympathetic neurons. Although CHT expression has been reported in noradrenergic sympathetic neurons of the superior cervical ganglion, these data indicate that in the sympathetic neurons projecting to sweat glands CHT is present at detectable levels only after association with the glands.     

Ophthalmoplegia due to mitochondrial DNA disease: the need for genetic diagnosis

We describe a patient with chronic progressive external ophthalmoplegia (CPEO) who underwent muscle biopsy for suspected mitochondrial disease. In spite of normal histocytochemical cytochrome c oxidase (COX) activity and respiratory chain enzyme measurements in muscle, subsequent molecular genetic analysis revealed the presence of a single, large-scale deletion of mitochondrial DNA (mtDNA). The case serves to illustrate the importance of pursuing the proposed mitochondrial genetic abnormality, even in patients with normal biopsy findings.