Altered brain structure with preserved cortical motor activity after exertional hypohydration: a MRI study.

Hypohydration exceeding 2% body mass can impair endurance capacity. It is postulated that the brain could be perturbed by hypohydration, leading to impaired motor performance. We investigated the neural effects of hypohydration with magnetic resonance imaging (MRI). Ten men were dehydrated to approximately -3% body mass by running on a treadmill at 65% maximal oxygen consumption (V̇o2max) before drinking to replace either 100% [euhydration (EU)] or 0% [hypohydration (HH)] of fluid losses. MRI was performed before start of trial (baseline) and after rehydration phase (post) to evaluate brain structure, cerebral perfusion, and functional activity. Endurance capacity assessed with a time-to-exhaustion run at 75% V̇o2max was reduced with hypohydration (EU: 45.2 ± 9.3 min, HH: 38.4 ± 10.7 min; P = 0.033). Mean heart rates were comparable between trials (EU: 162 ± 5 beats/min, HH: 162 ± 4 beats/min; P = 0.605), but the rate of rise in rectal temperature was higher in HH trials (EU: 0.06 ± 0.01°C/min, HH: 0.07 ± 0.02°C/min; P < 0.01). In HH trials, a reduction in total brain volume (EU: +0.7 ± 0.6%, HH: -0.7 ± 0.9%) with expansion of ventricles (EU: -2.7 ± 1.6%, HH: +3.7 ± 3.3%) was observed, and vice versa in EU trials. Global and regional cerebral perfusion remained unchanged between conditions. Functional activation in the primary motor cortex in left hemisphere during a plantar-flexion task was similar between conditions (EU: +0.10 ± 1.30%, HH: -0.11 ± 0.31%; P = 0.637). Our findings demonstrate that with exertional hypohydration, brain volumes were altered but the motor-related functional activity was unperturbed. NEW & NOTEWORTHY Dehydration occurs rapidly during prolonged or intensive physical activity, leading to hypohydration if fluid replenishment is insufficient to replace sweat losses. Altered hydration status poses an osmotic challenge for the brain, leading to transient fluctuations in brain tissue and ventricle volumes. Therefore, the amount of fluid ingestion during exercise plays a critical role in preserving the integrity of brain architecture. These structural changes, however, did not translate directly to motor functional deficits in a simple motor task.

Neural basis of exertional fatigue in the heat: A review of magnetic resonance imaging methods.

The central nervous system, specifically the brain, is implicated in the development of exertional fatigue under a hot environment. Diverse neuroimaging techniques have been used to visualize the brain activity during or after exercise. Notably, the use of magnetic resonance imaging (MRI) has become prevalent due to its excellent spatial resolution and versatility. This review evaluates the significance and limitations of various brain MRI techniques in exercise studies-brain volumetric analysis, functional MRI, functional connectivity MRI, and arterial spin labeling. The review aims to provide a summary on the neural basis of exertional fatigue and proposes future directions for brain MRI studies. A systematic literature search was performed where a total of thirty-seven brain MRI studies associated with exercise, fatigue, or related physiological factors were reviewed. The findings suggest that with moderate dehydration, there is a decrease in total brain volume accompanied with expansion of ventricular volume. With exercise fatigue, there is increased activation of sensorimotor and cognitive brain areas, increased thalamo-insular activation and decreased interhemispheric connectivity in motor cortex. Under passive hyperthermia, there are regional changes in cerebral perfusion, a reduction in local connectivity in functional brain networks and an impairment to executive function. Current literature suggests that the brain structure and function are influenced by exercise, fatigue, and related physiological perturbations. However, there is still a dearth of knowledge and it is hoped that through understanding of MRI advantages and limitations, future studies will shed light on the central origin of exertional fatigue in the heat.

Genetic Interactions Between TRPC6 and NPHS1 Variants Affect Posttransplant Risk of Recurrent Focal Segmental Glomerulosclerosis.

Individuals with TRPC6 mutations have variable phenotypes, ranging from healthy carrier to focal segmental glomerulosclerosis (FSGS) leading to renal failure. Here, we describe a family where six members had a novel TRPC6 p.R68W (c.202C>T) mutation, two of whom had renal failure from FSGS, and one had proteinuria. One healthy carrier donated a kidney to her sister. Both donor and recipient had no proteinuria at 20 years posttransplant. Two synonymous NPHS1 polymorphisms, rs2285450 (c.294C>T) and rs437168 (c.2289C>T) segregated with renal failure in this family. These variants had higher allele frequencies in 97 unrelated patients with nephrotic syndrome or FSGS compared to 224 controls. Using patch-clamp experiments in HEK293 and podocytes, we showed that the p.R68W mutation increased TRPC6 current amplitudes, which may be explained by enhanced TRPC6 surface expression. Additionally, while wild-type nephrin suppressed TRPC6 currents, this ability was lost in the presence of NPHS1 c.294C>T polymorphism. When cells were transfected according to combined TRPC6 and NPHS1 genotypes in the family, those representing the donor had lower TRPC6 currents than cells representing the recipient, suggesting that interactions between TRPC6 and NPHS1 variants could possibly account for the variable penetrance of TRPC6 mutations and the absence of recurrence in the graft.

Nuclear localization of Ca(v)2.2 and its distribution in the mouse central nervous system, and changes in the hippocampus during and after pilocarpine-induced status epilepticus.

AIMS: To investigate the subcellular localization of Ca(v)2.2 calcium channel in the mouse central nervous system (CNS), and changes of Ca(v)2.2 at acute and chronic stages during and after pilocarpine-induced status epilepticus (PISE), in order to find out the roles it may play in epileptogenesis. METHODS: Combined immunocytochemistry at both light and electron microscopic levels with real-time reverse transcription polymerase chain reaction (RT-PCR), cell transfection approach were used in this study. RESULTS: N-type calcium channel Ca(v)2.2 subunit was distributed in different regions of the mouse CNS. It was mainly localized in the nuclei in different types of neurones and in astrocytes. At acute stages during and after PISE, Ca(v)2.2 expression decreased in the stratum pyramidale of CA3 area and in the stratum granulosum of the dentate gyrus, but increased in the stratum lucidum of CA3 area and in the hilus of the dentate gyrus. At chronic stage at 2 months after PISE, increased expression of Ca(v)2.2 in both the strata granulosum and molecular of the dentate gyrus was observed. CONCLUSIONS: Ca(v)2.2 is a nuclear protein in neurones and astrocytes in the mouse CNS. Its translocation occurs at acute stages during and after PISE. The increased expression of Ca(v)2.2 in both the strata granulosum and moleculare of the dentate gyrus at chronic stage at 2 months after PISE may be involved in the occurrence of spontaneously recurrent seizures.

Age and gender-dependent alternative splicing of P/Q-type calcium channel EF-hand.

Ca(v)2.1 Ca(2+) channels (P/Q-type), which participate in various key roles in the CNS by mediating calcium influx, are extensively spliced. One of its alternatively-spliced exons is 37, which forms part of the EF hand. The expression of exon 37a (EFa form), but not exon 37b (EFb form), confers the channel an activity-dependent enhancement of channel opening known as Ca(2+)-dependent facilitation (CDF). In this study, we analyzed the trend of EF hand splice variant distributions in mouse, rat and human brain tissues. We observed a developmental switch in rodents, as well as an age and gender bias in human brain tissues, suggestive of a possible role of these EF hand splice variants in neurophysiological specialization. A parallel study performed on rodent brains showed that the data drawn from human and rodent tissues may not necessarily correlate in the process of aging.

Calmodulin bifurcates the local Ca2+ signal that modulates P/Q-type Ca2+ channels.

Acute modulation of P/Q-type (alpha1A) calcium channels by neuronal activity-dependent changes in intracellular Ca2+ concentration may contribute to short-term synaptic plasticity, potentially enriching the neurocomputational capabilities of the brain. An unconventional mechanism for such channel modulation has been proposed in which calmodulin (CaM) may exert two opposing effects on individual channels, initially promoting ('facilitation') and then inhibiting ('inactivation') channel opening. Here we report that such dual regulation arises from surprising Ca2+-transduction capabilities of CaM. First, although facilitation and inactivation are two competing processes, both require Ca2+-CaM binding to a single 'IQ-like' domain on the carboxy tail of alpha1A; a previously identified 'CBD' CaM-binding site has no detectable role. Second, expression of a CaM mutant with impairment of all four of its Ca2+-binding sites (CaM1234) eliminates both forms of modulation. This result confirms that CaM is the Ca2+ sensor for channel regulation, and indicates that CaM may associate with the channel even before local Ca2+ concentration rises. Finally, the bifunctional capability of CaM arises from bifurcation of Ca2+ signalling by the lobes of CaM: Ca2+ binding to the amino-terminal lobe selectively initiates channel inactivation, whereas Ca2+ sensing by the carboxy-terminal lobe induces facilitation. Such lobe-specific detection provides a compact means to decode local Ca2+ signals in two ways, and to separately initiate distinct actions on a single molecular complex.

Heterologous expression, functional characterization and localization of two isoforms of the monkey iron transporter Nramp2.

Natural resistance-associated macrophage protein 2 (Nramp2) has been suggested to be involved in transferrin-independent iron uptake. Two isoforms of the Nramp2 gene generated by alternative splicing of the 3' exons were identified in mouse, rat and human, but it is unclear if they perform distinct functions. To rationalize our previous work, which indicated an increase in iron deposition in a Parkinsonian monkey brain, two monkey Nramp2 isoforms were isolated for a comparative study to assess their relative iron-uptake abilities, tissue distribution and subcellular localization. The monkey Nramp2 isoforms, 2a and 2b, exhibit approx. 98% identity at the amino acid level when compared with the human homologues. The Nramp2a transcript contains a canonical iron-responsive element (IRE), whereas that of Nramp2b lacks the IRE motif in the 3' untranslated region. By reverse transcriptase (RT)-PCR, the mRNAs of both isoforms were detected in all tissues examined. The amino acid differences at the C-terminus neither affected the protein expression levels in HEK-293T and COS-7 cells nor altered the subcellular localization and tissue distribution of the isoforms. Similar levels of iron uptake were detected in the HEK-293T cells transfected with either the Nramp2a or 2b gene, and a reduction of iron from the ferric (Fe(3+)) to the ferrous (Fe(2+)) state is necessary before transport can take place. However, this transferrin-independent uptake of iron into the cells is not a Ca(2+)-dependent process.

Splicing of alpha 1A subunit gene generates phenotypic variants of P- and Q-type calcium channels.

P-type and Q-type calcium channels mediate neurotransmitter release at many synapses in the mammalian nervous system. The alpha 1A calcium channel has been implicated in the etiologies of conditions such as episodic ataxia, epilepsy and familial migraine, and shares several properties with native P- and Q-type channels. However, the exact relationship between alpha 1A and P- and Q-type channels is unknown. Here we report that alternative splicing of the alpha 1A subunit gene results in channels with distinct kinetic, pharmacological and modulatory properties. Overall, the results indicate that alternative splicing of the alpha 1A gene generates P-type and Q-type channels as well as multiple phenotypic variants.

The alpha 1E calcium channel exhibits permeation properties similar to low-voltage-activated calcium channels.

The physiological and pharmacological properties of the alpha 1E calcium (Ca) channel subtype do not exactly match any of the established categories described for native neuronal Ca currents. Many of the key diagnostic features used to assign cloned Ca channels to their native counterparts, however, are dependent on a number of factors, including cellular environment, beta subunit coexpression, and modulation by second messengers and G-proteins. Here, by examining the intrinsic pore characteristics of a family of transiently expressed neuronal Ca channels, we demonstrate that the permeation properties of alpha 1E closely resemble those described for a subset of low-threshold Ca channels. The alpha 1A (P-/Q-type), alpha 1B (N-type), and alpha 1C (L-type) high-threshold Ca channels all exhibit larger whole-cell currents with barium (Ba) as the charge carrier as compared with Ca or strontium (Sr). In contrast, macroscopic alpha 1E currents are largest in Sr, followed by Ca and then Ba. The unique permeation properties of alpha 1E are maintained at the single-channel level, are independent of the nature of the expression system, and are not affected by coexpression of alpha 2 and beta subunits. Overall, the permeation characteristics of alpha 1E are distinct from those described for R-type currents and share some similarities with native low-threshold Ca channels.

Beta subunit coexpression and the alpha1 subunit domain I-II linker affect piperidine block of neuronal calcium channels.

The effects of local anesthetics were examined on a family of transiently expressed neuronal calcium channels. Fomocaine, a local anesthetic containing a morpholine ring, preferentially blocked alpha1E channels (Ki = 100 microM), and had a lower affinity (3- to 15-fold) for alpha1A, alpha1B, and alpha1C channels. Block was incompletely reversible, followed 1:1 kinetics, and did not affect steady-state inactivation properties. Fomocaine block was sensitive to the concentration of permeant ion and enhanced in the presence of external pore blockers, suggesting a site of action in the conducting pathway. Flecainide, which carries a piperidine ring, and the diphenylbutylpiperidine antipsychotic, penfluridol, caused qualitatively similar block, suggesting that morpholine rings are compatible with the piperidine receptor site. In contrast, procaine, which contains an alkyl chain, caused reversible low affinity block of the different calcium channels (Kd values between 2 and 5 mM) and was least effective on alpha1E and did not compete with fomocaine, suggesting that local anesthetics interact with at least two distinct receptor sites. Compared to coexpression with the Ca channel beta1b subunit, block at the piperidine receptor site was significantly weakened with the beta2a subunit suggesting that the nature of the beta subunit contributes to drug binding. Amino acid changes in the cytoplasmic linker between domains I and II resulted in decreased fomocaine and penfluridol blocking affinity. Furthermore, the blocking affinity observed with alpha1B, was conferred on alpha1A by substitution of the domain I-II linker of alpha1B into alpha1A. Taken together, the data suggest that beta subunit binding and the domain I-II linker contribute to the piperidine receptor site on neuronal calcium channels.

Determinants of the G protein-dependent opioid modulation of neuronal calcium channels.

The modulation of a family of cloned neuronal calcium channels by stimulation of a coexpressed mu opioid receptor was studied by transient expression in Xenopus oocytes. Activation of the morphine receptor with the synthetic enkephalin [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO) resulted in a rapid inhibition of alpha1A (by approximately 20%) and alpha1B (by approximately 55%) currents while alpha1C and alpha1E currents were not significantly affected. The opioid-induced effects on alpha1A and alpha1B currents were blocked by pertussis toxin and the GTP analogue guanosine 5'-[beta-thio]diphosphate. Similar to modulation of native calcium currents, DAMGO induced a slowing of the activation kinetics and exhibited a voltage-dependent inhibition that was partially relieved by application of strong depolarizing pulses. alpha1A currents were still inhibited in the absence of coexpressed Ca channel alpha2 and beta subunits, suggesting that the response is mediated by the alpha1 subunit. Furthermore, the sensitivity of alpha1A currents to DAMGO-induced inhibition was increased approximately 3-fold in the absence of a beta subunit. Overall, the results show that the alpha1A (P/Q type) and the alpha1B (N type) calcium channels are selectively modulated by a GTP-binding protein (G protein). The results raise the possibility of competitive interactions between beta subunit and G protein binding to the alpha1 subunit, shifting gating in opposite directions. At presynaptic terminals, the G protein-dependent inhibition may result in decreased synaptic transmission and play a key role in the analgesic effect of opioids and morphine.

Essential Ca(2+)-binding motif for Ca(2+)-sensitive inactivation of L-type Ca2+ channels.

Intracellular calcium (Ca2+) inhibits the opening of L-type (alpha 1C) Ca2+ channels, providing physiological control of Ca2+ entry into a wide variety of cells. A structural determinant of this Ca(2+)-sensitive inactivation was revealed by chimeric Ca2+ channels derived from parental alpha 1C and alpha 1E channels, the latter of which is a neuronal channel lacking Ca2+ inactivation. A consensus Ca(2+)-binding motif (an EF hand), located on the alpha 1C subunit, was required for Ca2+ inactivation. Donation of the alpha 1C EF-hand region to the alpha 1E channel conferred the Ca(2+)-inactivating phenotype. These results strongly suggest that Ca2+ binding to the alpha 1C subunit initiates Ca2+ inactivation.

Biochemical properties and subcellular distribution of the neuronal class E calcium channel alpha 1 subunit.

Anti-peptide antibodies specific for the neuronal calcium channel alpha 1E subunit (anti-CNE1 and anti-CNE2) were produced to study the biochemical properties and subcellular distribution of the alpha 1E polypeptide from rat brain. Immunoblotting identified a single size form of 245-255 kDa which was a substrate for phosphorylation by cAMP-dependent protein kinase, protein kinase C, cGMP-dependent protein kinase, and calcium/calmodulin-dependent protein kinase II. Ligand-binding studies of alpha 1E indicate that it is not a high affinity receptor for the dihydropyridine isradipine or the peptide toxins omega-conotoxin GVIA or omega-conotoxin MVIIC at concentrations which elicit high affinity binding to other channel types in the same membrane preparation. The alpha 1E subunit is widely distributed in the brain with the most prominent immunocytochemical staining in deep midline structures such as caudate-putamen, thalamus, hypothalamus, amygdala, cerebellum, and a variety of nuclei in the ventral midbrain and brainstem. Staining is primarily in the cell soma but is also prominent in the dendritic field of a discrete subset of neurons including the mitral cells of the olfactory bulb and the distal dendritic branches of the cerebellar Purkinje cells. Our observations indicate that the 245-255 kDa alpha 1E subunit is localized in cell bodies, and in some cases in dendrites, of a broad range of central neurons and is potentially modulated by multiple second messenger-activated protein kinase.

Determinants of PKC-dependent modulation of a family of neuronal calcium channels.

The modulation of Ca2+ channel activity by protein kinases contributes to the dynamic regulation of neuronal physiology. Using the transient expression of a family of neuronal Ca2+ channels, we have identified several factors that contribute to the PKC-dependent modulation of Ca2+ channels. First, the nature of the Ca2+ channel alpha 1 subunit protein is critical. Both alpha 1B and alpha 1E channels exhibit a 30%-40% increase in peak currents after exposure to phorbol esters, whereas neither alpha 1A nor alpha 1C channels are significantly affected. This up-regulation can be mimicked for alpha 1E channels by stimulation of a coexpressed metabotropic glutamate receptor (type 1 alpha) through a PKC-dependent pathway. Second, PKC-stimulated up-regulation is dependent upon coexpression with a Ca2+ channel beta subunit. Third, substitution of the cytoplasmic domain I-II linker from alpha 1B confers PKC sensitivity to alpha 1A channels. The results provide direct evidence for the modulation of a subset of neuronal Ca2+ channels by PKC and implicate alpha 1 and beta subunit interactions in regulating channel activity via second messenger pathways.

Localization and functional properties of a rat brain alpha 1A calcium channel reflect similarities to neuronal Q- and P-type channels.

Functional expression of the rat brain alpha 1A Ca channel was obtained by nuclear injection of an expression plasmid into Xenopus oocytes. The alpha 1A Ca current activated quickly, inactivated slowly, and showed a voltage dependence typical of high voltage-activated Ca channels. The alpha 1A current was partially blocked (approximately 23%) by omega-agatoxin IVA (200 nM) and substantially blocked by omega-conotoxin MVIIC (5 microM blocked approximately 70%). Bay K 8644 (10 microM) or omega-conotoxin GVIA (1 microM) had no significant effect on the alpha 1A current. Coexpression with rat brain Ca channel beta subunits increased the alpha 1A whole-cell current and shifted the current-voltage relation to more negative values. While the beta 1b and beta 3 subunits caused a significant acceleration of the alpha 1A inactivation kinetics, the beta 2a subunit dramatically slowed the inactivation of the alpha 1A current to that seen typically for P-type Ca currents. In situ localization with antisense deoxyoligonucleotide and RNA probes showed that alpha 1A was widely distributed throughout the rat central nervous system, with moderate to high levels in the olfactory bulb, in the cerebral cortex, and in the CA fields and dentate gyrus of the hippocampus. In the cerebellum, prominent alpha 1A expression was detected in Purkinje cells with some labeling also in granule cells. Overall, the results show that alpha 1A channels are widely expressed and share some properties with both Q- and P-type channels.

Structure and functional expression of a member of the low voltage-activated calcium channel family.

Oscillatory firing patterns are an intrinsic property of some neurons and have an important function in information processing. In some cells, low voltage-activated calcium channels have been proposed to underlie a depolarizing potential that regulates bursting. The sequence of a rat brain calcium channel alpha 1 subunit (rbE-II) was deduced. Although it is structurally related to high voltage-activated calcium channels, the rbE-II channel transiently activated at negative membrane potentials, required a strong hyperpolarization to deinactivate, and was highly sensitive to block by nickel. In situ hybridization showed that rbE-II messenger RNA is expressed in regions throughout the central nervous system. The electrophysiological properties of the rbE-II current are consistent with a type of low voltage-activated calcium channel that requires membrane hyperpolarization for maximal activity, which suggests that rbE-II may be involved in the modulation of firing patterns.

Locus-specific transcriptional control of HLA genes.

One remarkable genetic feature of the class I MHC genes is their unparalleled degree of genetic polymorphism and diversity. The polymorphism is reflected by the fact that multiple loci encode class I molecules, and for each locus there are multiple alleles. In the course of investigating the regulation of HLA-A and HLA-B mRNA in human colorectal carcinoma cell lines, we have noticed a noncoordinate expression of the HLA mRNA in some of these cell lines. This observation prompted us to make use of these cell lines to study the locus-specific transcriptional regulation of HLA genes. Bandshift and footprint assays revealed at least three distinct and independent DNA-binding factors that bind to the core regulatory element of the HLA-A and HLB-B gene locus. A "novel" DNA-binding factor recognizing the CCAAT motif seems to be important for locus-specific expression of HLA-A mRNA, whereas a different factor which binds to a Sp1-like sequence is crucial for normal HLA-B mRNA expression. In certain colorectal cancer cell lines, underrepresentation of these locus-specific DNA-binding proteins correlates with the locus-specific down-regulation of HLA mRNA. This observation is further supported by experiments which demonstrated that the locus-specific suppression of exogenously introduced TK-chloramphenicol acetyltransferase DNA constructs, containing the "putative" HLA locus-specific DNA core regulatory sequence, is regulated in a locus-specific manner when introduced into these HLA-A- and HLA-B-deficient human colorectal cell lines.

HLA genotyping of colorectal carcinoma in the Chinese population.

The HLA-DR genotypes of 61 primary colorectal carcinomas obtained from patients of Chinese origin were determined by using DNA-RFLP. No increase or decrease of a particular HLA genotype could be ascertained with the disease, although we detected an antigen frequency of 29.5% for the serologically ill-defined DR"X3" specificity. We identified and sequenced HLA-DRB1 and DRB3 genes from the DR"X3" haplotype. The DR"X3" DRB1 gene was found to be identical to DRB1*1201 (DR5[w12]). A unique observation is its unusual linkage with DRB3*0101 (DRw52a) or DRB3*0301 (DRw52c) instead of the usual linkage with DRB3*0201/2 (DRw52b). These associations are rare in whites and blacks.

Regulation of HLA class I gene expression in human colorectal carcinoma.

The under-representation of HLA antigens in human tumors is usually associated with a poor prognosis. In this report, the expression of HLA class I genes in human colorectal carcinomas was studied using HLA-A and HLA-B locus-specific probes. Over 50% of the colorectal carcinomas studied showed a reduction in the amount of steady state HLA class I mRNA. For some carcinomas, non-coordinated regulation of the HLA-A and HLA-B genes was observed. The mechanism of HLA suppression was investigated and is most likely due to the presence of transcriptional regulatory elements.

Identification of locus-specific DNA-binding factors for the regulation of HLA class-I genes in human colorectal cancer.

The expression of HLA class-I mRNA in human colorectal cancer cell lines was studied. Locus-specific down-regulation of HLA class-I mRNA could be demonstrated in some of the human colorectal lines. This transcriptional suppression of HLA mRNA, however, was not a result of genetic alterations in the HLA structural genes. The transcription of the HLA class-I genes in the HLA-deficient cell lines could be induced by the addition of human recombinant gamma-IFN. In this report, we have employed these human colorectal cell lines to study locus-specific transcriptional regulation of HLA class-I gene expression. Our results demonstrate that the locus-specific suppression of HLA gene expression in human colorectal cell lines is mediated by the loss of certain DNA-binding transcription factors which act in a locus-specific manner. Our conclusion is further supported by experiments which showed that exogenously introduced HLA-CAT DNA constructs were also regulated in a locus-specific fashion when assayed by in vitro functional assays using these human colorectal cell lines.