Altered Notch Signaling in Developing Molar Teeth of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP)-Deficient Mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with neuroprotective and neurotrophic effects. This suggests its influence on the development of teeth, which are, similarly to the nervous system, ectoderm and neural crest derivatives. Our earlier studies have shown morphological differences between wild-type (WT) and PACAP-deficient mice, with upregulated sonic hedgehog (SHH) signaling in the lack of PACAP. Notch signaling is a key element of proper tooth development by regulating apoptosis and cell proliferation. In this study, our main goal was to evaluate the possible effects of PACAP on Notch signaling pathway. Immunohistochemical staining was performed of Notch receptors (Notch1, 2, 3, 4), their ligands [delta-like protein (DLL)1, 3, 4, Jagged1, 2], and intracellular target molecules [CSL (CBF1 humans/Su (H) Drosophila/LAG1 Caenorhabditis elegans transcription factor); TACE (TNF-α converting enzyme), NUMB] in molar teeth of 5-day-old WT, and homozygous and heterozygous PACAP-deficient mice. We measured immunopositivity in the enamel-producing ameloblasts and dentin-producing odontoblasts. Notch2 receptor and DLL1 expression were elevated in ameloblasts of PACAP-deficient mice compared to those in WT ones. The expression of CSL showed similar results both in the ameloblasts and odontoblasts. Jagged1 ligand expression was elevated in the odontoblasts of homozygous PACAP-deficient mice compared to WT mice. Other Notch pathway elements did not show significant differences between the genotype groups. The lack of PACAP leads to upregulation of Notch pathway elements in the odontoblast and ameloblast cells. The underlying molecular mechanisms are yet to be elucidated; however, we propose SHH-dependent and independent processes. We hypothesize that this compensatory upregulation of Notch signaling by the lack of PACAP could represent a salvage pathway in PACAP-deficient animals.

Alteration of the PAC1 Receptor Expression in the Basal Ganglia of MPTP-Induced Parkinsonian Macaque Monkeys.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a well-known neuropeptide with strong neurotrophic and neuroprotective effects. PACAP exerts its protective actions via three G protein-coupled receptors: the specific Pac1 receptor (Pac1R) and the Vpac1/Vpac2 receptors, the neuroprotective effects being mainly mediated by the Pac1R. The protective role of PACAP in models of Parkinson's disease and other neurodegenerative diseases is now well-established in both in vitro and in vivo studies. PACAP and its receptors occur in the mammalian brain, including regions associated with Parkinson's disease. PACAP receptor upregulation or downregulation has been reported in several injury models or human diseases, but no data are available on alterations of receptor expression in Parkinson's disease. The model closest to the human disease is the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced macaque model. Therefore, our present aim was to evaluate changes in Pac1R expression in basal ganglia related to Parkinson's disease in a macaque model. Monkeys were rendered parkinsonian with MPTP, and striatum, pallidum, and cortex were evaluated for Pac1R immunostaining. We found that Pac1R immunosignal was markedly reduced in the caudate nucleus, putamen, and internal and external parts of the globus pallidus, while the immunoreactivity remained unchanged in the cortex of MPTP-treated parkinsonian monkey brains. This decrease was attenuated in some brain areas in monkeys treated with L-DOPA. The strong, specific decrease of the PACAP receptor immunosignal in the basal ganglia of parkinsonian macaque monkey brains suggests that the PACAP/Pac1R system may play an important role in the development/progression of the disease.

Chronic stress-induced mechanical hyperalgesia is controlled by capsaicin-sensitive neurones in the mouse.

BACKGROUND: Clinical studies demonstrated peripheral nociceptor deficit in stress-related chronic pain states, such as fibromyalgia. The interactions of stress and nociceptive systems have special relevance in chronic pain, but the underlying mechanisms including the role of specific nociceptor populations remain unknown. We investigated the role of capsaicin-sensitive neurones in chronic stress-related nociceptive changes. METHOD: Capsaicin-sensitive neurones were desensitized by the capsaicin analogue resiniferatoxin (RTX) in CD1 mice. The effects of desensitization on chronic restraint stress (CRS)-induced responses were analysed using behavioural tests, chronic neuronal activity assessment in the central nervous system with FosB immunohistochemistry and peripheral cytokine concentration measurements. RESULTS: Chronic restraint stress induced mechanical and cold hypersensitivity and increased light preference in the light-dark box test. Open-field and tail suspension test activities were not altered. Adrenal weight increased, whereas thymus and body weights decreased in response to CRS. FosB immunopositivity increased in the insular cortex, dorsomedial hypothalamic and dorsal raphe nuclei, but not in the spinal cord dorsal horn after the CRS. CRS did not affect the cytokine concentrations of hindpaw tissues. Surprisingly, RTX pretreatment augmented stress-induced mechanical hyperalgesia, abolished light preference and selectively decreased the CRS-induced neuronal activation in the insular cortex. RTX pretreatment alone increased the basal noxious heat threshold without influencing the CRS-evoked cold hyperalgesia and augmented neuronal activation in the somatosensory cortex and interleukin-1α and RANTES production. CONCLUSIONS: Chronic restraint stress induces hyperalgesia without major anxiety, depression-like behaviour or peripheral inflammatory changes. Increased stress-induced mechanical hypersensitivity in RTX-pretreated mice is presumably mediated by central mechanisms including cortical plastic changes. SIGNIFICANCE: These are the first data demonstrating the complex interactions between capsaicin-sensitive neurones and chronic stress and their impact on nociception. Capsaicin-sensitive neurones are protective against stress-induced mechanical hyperalgesia by influencing neuronal plasticity in the brain.

Age-related changes in acute central leptin effects on energy balance are promoted by obesity.

Leptin is a key catabolic regulator of food intake (FI) and energy expenditure. Both aging and obesity have been shown to induce leptin-resistance. The present study aimed to analyze age-related changes in the anorexigenic and hypermetabolic responsiveness to acute intracerebroventricular leptin administration in different age-groups of normally fed male Wistar rats (adult and old rats from 3 to 24months of age, NF3 to NF24, respectively). The expressions of the long form of the leptin receptor (Ob-Rb) and inhibitory SOCS3 genes were also assessed by quantitative RT-PCR in the arcuate nucleus (ARC). The influence of high-fat diet-induced obesity (HF) on the anorexigenic leptin effects were also tested in younger and older middle-aged groups (HF6 and HF12). Leptin-induced anorexia varied with age: leptin suppressed re-feeding FI (following 48-h fasting) strongly in young adult (NF3), but not in younger or older middle-aged (NF6 or NF12) or in aging (NF18) rats. However, anorexigenic leptin effects reached statistical significance again in old NF24 rats. Leptin-induced hypermetabolism, on the other hand, showed monotonous age-related decline and disappeared by old age. Ob-Rb expression declined until 12months of age followed by a partial recovery in NF18 and NF24 groups. On the other hand, SOCS3 expression was high in NF6 and NF18 and to some extent in NF24 rats. Age-related alterations of Ob-Rb and SOCS3 expression in the ARC may partly contribute to the explanation of age-related variations in anorexigenic but not hypermetabolic leptin effects. High-fat diet-induced obesity was associated with resistance to leptin-induced anorexia in HF6, similar to that seen in NF6. However, instead of the expected leptin-resistance in HF12, a strong leptin-induced suppression of re-feeding was detected in these obese middle-aged rats. Our results suggest that acute central effects of leptin on anorexia and hypermetabolism change in disparate ways during aging, implying separate mechanisms (e.g. signal transduction pathways) of different leptin actions. The age-related pattern shown by leptin-induced anorexia may contribute to the explanation of middle-aged obesity, and partly to that of aging anorexia. Our findings concerning obese rats are in accord with previous observations on anorexigenic effects of peripherally administered cholecystokinin: diet-induced obesity appeared to accelerate the development of age-related regulatory alterations. Similarly, our present data also raise the possibility that chronic diet-induced obesity promotes responsiveness to centrally applied leptin at least concerning anorexigenic effects.

Comparison of arterial stiffness parameters in patients with coronary artery disease and diabetes mellitus using Arteriograph.

Recently an expert consensus document advised to standardize user procedures and a new cut-off value for carotid-femoral pulse wave velocity in daily practice. Our aim was to observe aortic pulse wave velocity (PWVao) and augmentation index (AIXao) in two high cardiovascular risk groups: patients with verified coronary artery disease (CAD) or with type 2 diabetes mellitus (T2DM). We also aimed to determine the cut-off values for PWVao, AIXao in CAD and T2DM patients using oscillometric device (Arteriograph). We investigated 186 CAD and 152 T2DM patients. PWVao and AIXao increased significantly in the CAD group compared to the age-, gender-, blood pressure-, and heart rate-matched control group (10.2+/-2.3 vs. 9.3+/-1.5 m/s; p<0.001 and 34.9+/-14.6 vs. 31.9+/-12.8 %; p<0.05, respectively). When compared to the apparently healthy control subjects, T2DM patients had significantly elevated PWVao (9.7+/-1.7 vs. 9.3+/-1.5 m/s; p<0.05, respectively), however the AIXao did not differ significantly. The ROC-curves of CAD and healthy control subjects explored cut-off values of 10.2 m/s for PWVao and 33.23 % for AIXao. Our data provide supporting evidence about impaired arterial stiffness parameters in CAD and T2DM. Our findings encourage the implementation of arterial stiffness measurements by oscillometric method in daily clinical routine.

Comparative protein composition of the brains of PACAP-deficient mice using mass spectrometry-based proteomic analysis.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a widespread neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. The diverse biological actions provide the background for the variety of deficits observed in mice lacking endogenous PACAP. PACAP-deficient mice display several abnormalities, such as sudden infant death syndrome (SIDS)-like phenotype, decreased cell protection, and increased risk of Parkinson's disease. However, the molecular and proteomic background is still unclear. Therefore, our aim was to investigate the differences in peptide and protein composition in the brains of PACAP-deficient and wild-type mice using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric (MS)-based proteomic analysis. Brains from PACAP-deficient mice were removed, and different brain areas (cortex, hippocampus, diencephalon, mesencephalon, brainstem, and cerebellum) were separated. Brain pieces were weighed, homogenized, and further processed for electrophoretic analysis. Our results revealed several differences in diencephalon and mesencephalon. The protein bands of interest were cut from the gel, samples were digested with trypsin, and the tryptic peptides were measured by matrix-assisted laser desorption ionization time of flight (MALDI TOF) MS. Results were analyzed by MASCOT Search Engine. Among the altered proteins, several are involved in metabolic processes, energy homeostasis, and structural integrity. ATP-synthase and tubulin beta-2A were expressed more strongly in PACAP-knockout mice. In contrast, the expression of more peptides/proteins markedly decreased in knockout mice, like pyruvate kinase, fructose biphosphate aldolase-A, glutathione S-transferase, peptidyl propyl cis-trans isomerase-A, gamma enolase, and aspartate amino transferase. The altered expression of these enzymes might partially account for the decreased antioxidant and detoxifying capacity of PACAP-deficient mice accompanying the increased vulnerability of these animals. Our results provide novel insight into the altered biochemical processes in mice lacking endogenous PACAP.

PACAP immunoreactivity in human malignant tumor samples and cardiac diseases.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic and multifunctional neuropeptide having important roles in various physiological processes. Recent trends in PACAP research point to the clinical introduction of PACAP or its analogs/fragments possibly in the near future. Recently, we have shown the presence of PACAP in human plasma, milk, placenta, and follicular fluid samples. However, relatively few data are available on PACAP in human tissues from patients with different disorders. The aim of the present study was to determine, by radioimmunoassay, the tissue level of PACAP38-like immunoreactivity (LI) and PACAP27-LI in different primary non-small cell lung cancer, colon tumor samples, and in cardiac muscle samples from patients suffering from ischemic heart disease and valvular disorders. We also labeled the PAC1 receptors in human cardiac cells. All samples showed significantly higher PACAP38-LI compared with PACAP27-LI. We found significantly lower levels of PACAP38-LI and PACAP27-LI in tumoral and peripheral samples compared with normal healthy tissue in both lung and colon cancers. Further investigations are necessary to describe the exact function of PACAP in oncogenesis. We showed that PACAP38-LI and PACAP27-LI are significantly higher in ischemic heart diseases compared with valvular abnormalities, suggesting that PACAP might play a role in ischemic heart disorders.

The behavioral phenotype of pituitary adenylate-cyclase activating polypeptide-deficient mice in anxiety and depression tests is accompanied by blunted c-Fos expression in the bed nucleus of the stria terminalis, central projecting Edinger-Westphal nucleus, ventral lateral septum, and dorsal raphe nucleus.

Pituitary adenylate-cyclase activating polypeptide (PACAP) has been implicated in the (patho)physiology of stress-adaptation. PACAP deficient (PACAP(-/-)) mice show altered anxiety levels and depression-like behavior, but little is known about the underlying mechanisms in stress-related brain areas. Therefore, we aimed at investigating PACAP(-/-) mice in light-dark box, marble burying, open field, and forced swim paradigms. We also analyzed whether the forced swim test-induced c-Fos expression would be affected by PACAP deficiency in the following stress-related brain areas: magno- and parvocellular paraventricular nucleus of the hypothalamus (PVN); basolateral (BLA), medial (MeA), and central (CeA) amygdaloid nuclei; ventral (BSTv), dorsolateral (BSTdl), dorsomedial (BSTdm), and oval (BSTov) nuclei of the bed nucleus of stria terminalis; dorsal (dLS) and ventral parts (vLS) of lateral septal nucleus, central projecting Edinger-Westphal nucleus (EWcp), dorsal (dPAG) and lateral (lPAG) periaqueductal gray matter, dorsal raphe nucleus (DR). Our results revealed that PACAP(-/-) mice showed greatly reduced anxiety and increased locomotor activity compared with wildtypes. In forced swim test PACAP(-/-) mice showed increased depression-like behavior. Forced swim exposure increased c-Fos expression in all examined brain areas in wildtypes, whereas this was markedly blunted in the DR, EWcp, BSTov, BSTdl, BSTv, PVN, vLS, dPAG, and in the lPAG of PACAP(-/-) mice vs. wildtypes, strongly suggesting their involvement in the behavioral phenotype of PACAP(-/-) mice. PACAP deficiency did not influence the c-Fos response in the CeA, MeA, BSTdm, and dLS. Therefore, we propose that PACAP exerts a brain area-specific effect on stress-induced neuronal activation and it might contribute to stress-related mood disorders.

Differential responses of corticotropin-releasing factor and urocortin 1 to acute pain stress in the rat brain.

It has been hypothesized that corticotropin-releasing factor (CRF) and its related neuropeptide urocortin 1 (Ucn1) play different roles in the initiation and adaptive phases of the stress response, which implies different temporal dynamics of these neuropeptides in response to stressors. We have tested the hypothesis that acute pain stress (APS) differentially changes the dynamics of CRF expression in the paraventricular nucleus of the hypothalamus (PVN), oval subdivision of the bed nucleus of the stria terminalis (BSTov) and central amygdala (CeA), and the dynamics of Ucn1 expression in the midbrain non-preganglionic Edinger-Westphal nucleus (npEW). Thirty minutes after APS, induced by a formalin injection into the left hind paw, PVN, BSTov, CeA and npEW all showed a peak in cFos mRNA expression that was followed by a robust increase in cFos protein-immunoreactivity, indicating a rapid increase in (immediate early) gene expression in all four brain nuclei. CRF-dynamics, however, were affected by APS in a brain nucleus-specific way: in the PVN, CRF-immunoreactivity was minimal at 60 min after APS and concomitant with a marked increase in plasma corticosterone, whereas in the BSTov not CRF peptide but CRF mRNA peaked at 60 min, and in the CeA a surge of CRF peptide occurred as late as 240 min. The npEW differed from the other centers, as Ucn1 mRNA and Ucn1 peptide peaked at 120 min. These results support our hypothesis that each of the four brain centers responds to APS with CRF/Ucn1 dynamics that are specific as to nature and timing. In particular, we propose that CRF in the PVN plays a major role in the initiation phase, whereas Ucn1 in the npEW may act in the later, termination phase of the adaptation response to APS.

Selective association of endogenous ouabain with subclinical organ damage in treated hypertensive patients.

According to previous studies endogenous ouabain (EO) closely correlates with high blood pressure, congestive heart failure and kidney disease in humans. Our aims were to analyse associations between plasma, urinary EO level and various markers of cardiovascular damage in treated hypertensive patients. Forty-one adult patients with hypertension and/or diabetes mellitus (DM) and/or chronic kidney disease (CKD) were studied. We assessed plasma and urinary EO, pro-brain natriuretic peptide and catecholamines, profile of ambulatory blood pressure monitor and cardiovascular status by echocardiography and echo-tracking. The highest level of plasma EO (19.7±9.5 pmol l⁻¹) was measured in hypertensive patients with DM and CKD. The nighttime mean arterial blood pressure independently correlated with the level of plasma EO (P=0.004), while independent predictor of the ß-stiffness of carotid artery was the urinary EO (P=0.011). Elevated level of EO was associated with nighttime blood pressure and subclinical organ damage in treated hypertensive patients, suggesting possible role of EO in the pathogenesis of impaired diurnal blood pressure rhythm and arterial stiffness.

Impaired nocifensive behaviours and mechanical hyperalgesia, but enhanced thermal allodynia in pituitary adenylate cyclase-activating polypeptide deficient mice.

Pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38) and its receptors (PAC1 and VPAC) have been shown in the spinal dorsal horn, dorsal root ganglia and sensory nerve terminals. Data concerning the role of PACAP in central pain transmission are controversial and we have recently published its divergent peripheral effects on nociceptive processes. The aim of the present study was to investigate acute somatic and visceral nocifensive behaviours, partial sciatic nerve ligation-evoked chronic neuropathic, as well as resiniferatoxin-induced inflammatory thermal and mechanical hyperalgesia in PACAP deficient (PACAP(-/-)) mice to elucidate its overall function in pain transmission. Neuronal activation was investigated with c-Fos immunohistochemistry. Paw lickings in the early (0-5 min) and late (20-45 min) phases of the formalin test were markedly reduced in PACAP(-/-) mice. Acetic acid-evoked abdominal contractions referring to acute visceral chemonociception was also significantly attenuated in PACAP knockout animals. In both models, the excitatory role of PACAP was supported by markedly greater c-Fos expression in the periaqueductal grey and the somatosensory cortex. In PACAP-deficient animals neuropathic mechanical hyperalgesia was absent, while c-Fos immunopositivity 20 days after the operation was significantly higher. In this chronic model, these neurons are likely to indicate the activation of secondary inhibitory pathways. Intraplantarly injected resiniferatoxin-evoked mechanical hyperalgesia involving both peripheral and central processes was decreased, but thermal allodynia mediated by only peripheral mechanisms was increased in PACAP(-/-) mice. These data clearly demonstrate an overall excitatory role of PACAP in pain transmission originating from both exteroceptive and interoceptive areas, it is also involved in central sensitization. This can be explained by the signal transduction mechanisms of its identified receptors, both PAC1 and VPAC activation leads to neuronal excitation. In contrast, it is an inhibitory mediator at the level of the peripheral sensory nerve endings and decreases their sensitization to heat with presently unknown mechanisms.

Stress-related changes in the activity of cocaine- and amphetamine-regulated transcript and nesfatin neurons in the midbrain non-preganglionic Edinger-Westphal nucleus in the rat.

Cocaine- and amphetamine-regulated transcript (CART) and nesfatin-1/nucleobindin 2 (NUCB2) are assumed to play a role in feeding and adaptation to stress. Both peptides are highly expressed in the midbrain non-preganglionic Edinger-Westphal nucleus (npEW), a center implicated in the regulation of stress adaptation and in the pathogenesis of stress-induced brain disorders, in a sex-specific manner. The present study was undertaken to test whether CART and nesfatin are involved in these actions of the npEW in the rat. Acute restraint and chronic variable mild stress were used. Following stress, physiological parameters (serum corticosterone levels, body, adrenal and thymus weights) were determined, CART and nesfatin-like immunoreactivity (LI) as well as mRNA expression were analyzed in the npEW nucleus. Our results depict the following changes: (1) Acute stress resulted in an increase in serum corticosterone levels that was higher in females; (2) In males, data on corticosterone and body weight gain and in females, data on body weight gain revealed an effect of chronic stress; (3) Both acute and chronic stress activated npEW neurons expressing CART and nesfatin-LI, as shown by increased cFos immunoreactivity; (4) Chronic, but not acute stress increased the amount of CART and nesfatin-LI in both males and females; (5) Neither acute nor chronic stress had an effect on CART and NUCB2 mRNA contents of npEW neurons in either sex. Taken together, our data suggest that CART and nesfatin are involved in the response of npEW neurons to chronic stress.

Sex-specific effects of fasting on urocortin 1, cocaine- and amphetamine-regulated transcript peptide and nesfatin-1 expression in the rat Edinger-Westphal nucleus.

Leptin is critical for normal food intake and energy metabolism. While leptin receptor (ObR) function has been well studied in hypothalamic feeding circuitries, the functional relevance of ObR in extrahypothalamic areas is largely unknown. Central regulatory pathways involved in food intake utilize various neuropeptides, such as urocortin 1 (Ucn1), cocaine- and amphetamine-regulated transcript peptide (CART) and nesfatin-1. Ucn1 is most abundantly expressed in the non-preganglionic Edinger-Westphal nucleus (npEW). In addition to Ucn1, other satiety signals, such as CART and nesfatin-1, are highly expressed in neurons of the npEW. Using immunocytochemistry and reverse transcriptase polymerase chain reaction (RT-PCR), we here show the presence of short and long forms of ObR in the rat npEW. Then, we tested our hypothesis that a change in plasma leptin will modulate the activity of npEW neurons containing Ucn1, CART and nesfatin-1. First, by double-labeling immunocytochemistry, we observed that almost all npEW neurons colocalizing Ucn1, CART and nesfatin-1 also contain ObR. Fasting rats for two days caused a marked body weight loss and reduced leptin plasma level in both genders. Ucn1 mRNA and CART mRNA were upregulated after fasting in males (3.3 and 2.4 times, respectively; P<0.05) but not in females. However, their peptide levels were not significantly changed. The peptide level and mRNA of nesfatin-1 were unaffected by fasting. We conclude that npEW-neurons containing Ucn1, CART and nesfatin-1 co-express ObR, and may be involved in leptin-mediated feeding control in male rats only.

Diurnal expression of period 2 and urocortin 1 in neurones of the non-preganglionic Edinger-Westphal nucleus in the rat.

Period 2 (Per2) is an important clock gene involved in the regulation of the major circadian clock in the mammalian central nervous system, the suprachiasmatic nucleus. In addition, Per2 is expressed in many other stress-sensitive brain structures. We have previously showed that the non-preganglionic Edinger-Westphal nucleus (npEW) is the main site of the corticotropin-releasing factor peptide family member urocortin 1 (Ucn1) and that this peptide undergoes conspicuous expression changes in response to various stressors. Here, we hypothesized that in the rat npEW both Per2 and Ucn1 would be produced in a diurnal, rhythmical fashion. This hypothesis was tested by following this expected rhythm on two days in rats killed at four time points each day (Zeitgeber times 0, 6, 12, and 18). We showed the co-existence of Per2 and Ucn1 in the npEW with double-label immunofluorescence and demonstrated with quantitative RT-PCR and semi-quantitative immunocytochemistry diurnal rhythms in Per2 mRNA expression and Per2 protein content, each on a single different day, with a minimum at lights-off and a maximum at lights-on. We furthermore revealed a diurnal rhythm in the number of Ucn1-immunopositive neurones and in their Ucn1 peptide content, with a minimum at night and at the beginning of the light period and a peak at lights-off, while the Ucn1 mRNA content paralleled the Per2 mRNA rhythm. The rhythms were accompanied by a diurnal rhythm in plasma corticosterone concentration. Our results are in line with the hypothesis that both Per2 and Ucn1 in the rat npEW are produced in a diurnal fashion, a phenomenon that may be relevant for the regulation of the diurnal rhythm in the stress response.

Housekeeping genes revisited: different expressions depending on gender, brain area and stressor.

Housekeeping gene (HKG) mRNAs are used to normalize expression data of genes of interest in quantitative reverse transcriptase polymerase chain reaction studies. Such normalization assumes constant HKG gene expression under all circumstances. Although sporadic evidence suggests that HKG expression may not always fulfill this requirement and, therefore, such normalization may lead readily to erroneous results, this fact is generally not sufficiently appreciated by investigators. Here, we have systematically analyzed the expression of three common HKGs, glyceraldehyde-3-phosphate dehydrogenase, ribosomal subunit 18S and beta-actin, in two different stress paradigms, in various brain areas, in male and in female rats. HKG expressions differed considerably with respect to brain area, type of stressor and gender, in an HKG-specific manner. Therefore, we conclude that before final experimentation, pilot expression studies are necessary to select an HKG which expression is unaffected by the experimental factor(s), allowing reliable interpretation of expression data of genes of interest.

Effect of PACAP in 6-OHDA-induced injury of the substantia nigra in intact young and ovariectomized female rats.

Pituitary adenylate cyclase activating polypeptide (PACAP) has neuroprotective effects in various neuronal cultures and in models of brain pathologies in vivo. Among others, it protects dopaminergic neurons in vitro, against 6-OHDA- and rotenone-induced injury. Recently, we have shown that PACAP reduces dopaminergic cell loss and ameliorates behavioral outcome following unilateral 6-OHDA-induced injury of the substantia nigra in male rats. However, after castration, PACAP led only to a slight amelioration of the behavioral symptoms. The aim of the present study was to investigate the degree of neuroprotection exerted by PACAP in female rats, using the same model. It was found that PACAP had no effect on the dopaminergic cell loss in intact female rats, only caused amelioration of certain acute behavioral signs. In contrast, PACAP effectively increased dopaminergic cell survival and decreased behavioral deficits in ovariectomized females. These results indicate that the neuroprotective effect of PACAP in a rat model of Parkinson's disease is gender-specific.

The effects of the NMR shift-reagents Dy(PPP)2, Dy(TTHA) and Tm(DOTP) on developed pressure in isolated perfused rat hearts. The role of shift-reagent calcium complexes.

The 23Na NMR shift-reagent complexes (Dy(PPP)2, Dy(TTHA), and Tm(DOTP)) bind stoichiometric amounts of Ca2+. Thus, in perfused rat heart systems, a supplementation of Ca2+ is required to maintain the requisite extracellular free calcium concentration ([Ca(o)]f) and to approximate a physiological level of contractile function. The amount of reagent-bound Ca2+ in a heart perfusate that contains a shift-reagent depends on: (1) Ca2+ binding by excess ligand used during the preparation of the shift-reagent; and (2) the Ca2+ binding affinity of the shift-reagent. To address point 1), we introduced a 1H and 31P NMR spectroscopic titration method to quantify directly the concentration of the excess ligand. We also used this method to minimize the amount of excess ligand (L) and thus the amount of Ca*L complex. To address point (2), we determined the stepwise Kd (microm) values of the Ca complexes of the three shift-reagents.: Dy(PPP)2, Kd=0.09, Kd2=7.9; Dy(TTHA), Kd1=10.66, Kd2=10.12; and Tm(DOTP), K(d1)=0.502, Kd2=4.98. The Kd values of the Ca complexes of the phosphonate and triphosphate based shift-reagents, Tm(DOTP) and Dy(PPP)2, respectively, are lower than those of the polyaminocarboxylate-based Dy(TTHA), indicating stronger Ca binding affinities for the former two types of complexes. We have also shown a positive correlation between [Ca(o)]f and left ventricular developed pressure (LVDP) in perfused rat hearts. Dy(TTHA) has shown no effect on LVDP v[Ca(o)]f. The LVDP values in the presence of the phosphonate and triphosphate based shift-reagents, however, were significantly higher than expected from the [Ca(o)]f levels alone. Thus a positive inotropic effect, independent of [Ca(o)]f, is evident in the presence of Tm(DOTP) or Dy(PPP)2.

Replacement of ATP with ADP affects the dynamic and conformational properties of actin monomer.

The effect of the replacement of ATP with ADP on the conformational and dynamic properties of the actin monomer was investigated, by means of electron paramagnetic resonance (EPR) and fluorescence spectroscopic methods. The measurement of the ATP concentration during these experiments provided the opportunity to estimate the time dependence of ADP-Mg-G-actin concentration in the samples. According to the results of the fluorescence resonance energy transfer experiments, the Gln-41 and Cys-374 residues are closer to each other in the ADP-Mg-G-actin than in the ATP-Mg-G-actin. The fluorescence resonance energy transfer efficiency increased simultaneously with the ADP-G-actin concentration and reached its maximum value within 30 min at 20 degrees C. The EPR data indicate the presence of an ADP-Mg-G-actin population that can be characterized by an increased rotational correlation time, which is similar to the one observed in actin filaments, and exists only transiently. We suggest that the conformational transitions, which were reflected by our EPR data, were coupled with the transient appearance of short actin oligomers during the nucleotide exchange. Besides these relatively fast conformational changes, there is a slower conformational transition that could be detected several hours after the initiation of the nucleotide exchange.

23Na NMR shift reagents enhance cardiac staircase effect in isolated perfused rat hearts.

The effects of the currently used (23)Na NMR shift reagents, dysprosium bis-triphosphate [Dy(PPP)(2)], dysprosium triethylenetriamine hexaacetate [Dy(TTHA)] and thulium 1,4,7, 10-tetraazacyclododecane-N,N',N",N"'-tetra(methylenephosphonate) [Tm(DOTP)] were studied in the rat heart cardiac staircase model. Rat hearts were perfused with low or normal extracellular free calcium ([Ca(o)](f)). At low [Ca(o)](f) (0.34 +/- 0.05 mM), hearts were perfused with Dy(PPP)(2) (group I), Dy(TTHA) (group II) or no shift reagent (group III), while at normal [Ca(o)](f) (1.25 +/- 0.15 mM), hearts were perfused with Tm(DOTP) (group IV), Dy(TTHA) (group V) or no shift reagent (group VI). Left ventricular developed pressure (LVDP) values in group I were significantly higher than in groups II and III (p < 0.01), while no significant differences were found between groups II and III. LVDP values in group IV were significantly higher than in groups V and VI (p < 0.05), while the LVDP values in groups V and VI were almost identical. Also, a positive correlation between pacing rate and intracellular sodium ([Na(i)]) was evident. The [Na(i)] values at high [Ca(o)](f) were significantly lower than at low [Ca(o)](f) at each pacing level (p <0.01), indicating a negative correlation between [Na(i)] and [Ca(o)](f). No statistical differences were found in [Na(i)] between groups I vs II and IV vs V, showing that determination of [Na(i)] is not affected by any of these shift reagents. Thus the different LVDP responses in groups I vs II and IV vs V were not mirrored in [Na(i)] changes. We hypothesize that a direct, sarcolemmal Ca-Dy(PPP)(2)-, or Ca-Tm(DOTP)-induced positive inotropic effect could be responsible for these Na(i)-independent LVDP increases in groups I and IV.

Synthesis and in vivo evaluation of new contrast agents for cardiac MRI.

Analogues 2-6 of N(3),N(6)-bis(2'-myristoyloxyethyl)-1, 8-dioxotriethylenetetraamine-N,N,N',N'-tetraacetic acid (BME-DTTA) (1), which like 1 are also based on the DTTA structure but contain shorter fatty acyl chains, were synthesized to improve the water solubility of the corresponding gadolinium complexes. The gadolinium complexes of 1 and 3-5 have very low solubility in water. Thus liposomal preparations are necessary for their in vivo MRI application. These liposomal preparations retain high in vitro relaxivities (27.1, 21.57, 20.32, 23.1 s(-1) mM(-1), respectively) and induce sustained MRI signal intensity enhancements (67.2, 38.4, 52.1, 41.7 in arbitrary units, respectively). The gadolinium complex of 2 is quite soluble in water. Its lifetime in the blood stream, however, is short. The gadolinium complex of analogue 6, N-(2-butyryloxyethyl)-N'-(2-ethyloxyethyl)-N,N'-bis[N' ',N' '-bis(carboxymethyl)acetamido]-1,2-ethanediamine (ABE-DTTA), has demonstrated its potential as a water-soluble, cardiac-specific, MRI contrast agent. It is completely soluble in water at a 25 mM concentration, allowing the preparation of an injectable dose. The in vitro relaxivity of the complex is 16.24 s(-1) mM(-1). The agent shows a specific accumulation in the heart tissue reaching its maximum within 15 min after administration, inducing a sustained MRI signal intensity enhancement of 43.6%. This enhancement lasts for at least 3 h, thus indicating a reasonably long lifetime of this contrast agent in the myocardium without deleterious effects on heart function parameters.