Shinji Morita

Targeted Disruption of the Tyrosine Hydroxylase Locus Results in Severe Catecholamine Depletion and Perinatal Lethality in Mice

Tyrosine 3-hydroxylase (TH, EC 1.14.16.2) catalyzes the first and rate-limiting step of the catecholamine biosynthetic pathway in the nervous and endocrine systems. The TH locus was disrupted in mouse embryonic stem cells by homologous recombination. Mice heterozygous for the TH mutation were apparently normal. In these mice, TH activity in the embryos and adult tissues was less than 50% of the wild-type values, but the catecholamine level was decreased only moderately in the developing animals and was maintained normally at adulthood, suggesting the presence of a regulatory mechanism for ensuring the proper catecholamine level during animal development. In contrast, the homozygous mutant mice died at a late stage of embryonic development or shortly after birth. Both TH mRNA and enzyme activity were lacking in the homozygous mutants, which thus explained the severe depletion of catecholamines. These changes, however, did not affect gross morphological development of the cells that normally express high catecholamine levels. Analysis of electrocardiograms of surviving newborn mutants showed bradycardia, suggesting an alteration of cardiac functions in the homozygous mice that may lead to the lethality of this mutation. In addition, transfer of a human TH transgene into the homozygous mice corrected the mutant phenotype, showing recovery of TH activity by expression of the human enzyme. These results indicate that TH is essential for survival of the animals during the late gestational development and after birth.

Alteration of Catecholamine Phenotype in Transgenic Mice Influences Expression of Adrenergic Receptor Subtypes

Abstract: Agonist‐induced regulation of adrenergic receptors (ARs) has an important role in controlling physiological functions in response to changes in catecholamine stimulation. We previously generated transgenic mice expressing phenylethanolamine N‐methyltransferase (PNMT) under the control of a human dopamine β‐hydroxylase gene promoter to switch catecholamine specificity from the norepinephrine phenotype to the epinephrine phenotype. In the present study, we first examined changes in catecholamine metabolism in peripheral tissues innervated by sympathetic neurons of the transgenic mice. In the transgenic target tissues, a high‐level expression of PNMT led to a dramatic increase in the epinephrine levels, whereas the norepinephrine levels were decreased to 48.6–87.9% of the nontransgenic control levels. Analysis of plasma catecholamines in adrenalectomized mice showed large amounts of epinephrine derived from sympathetic neurons in the transgenic mice. Subsequently, we performed radioligand binding assays with (−)‐[125I]iodocyanopindolol to determine changes in binding sites of β‐AR subtypes. In transgenic mice, the number of β2‐AR binding sites was 56.4–74.9% of their nontransgenic values in the lung, spleen, submaxillary gland, and kidney, whereas the β1‐AR binding sites were regulated in a different fashion among these tissues. Moreover, northern blot analysis of total RNA from the lung tissues showed that down‐regulation of β2 binding sites was accompanied by a significant decrease in steady‐state levels of the receptor mRNA. These results strongly suggest that alteration of catecholamine specificity in the transgenic sympathetic neurons leads to regulated expression of the β‐AR subtypes in their target tissues.

Nicotine-induced regulation of tyrosine hydroxylase activity in adrenal gland of transgenic mouse carrying human tyrosine hydroxylase gene

We investigated the effect of subcutaneous injection of nicotine on in vitro tyrosine hydroxylase (TH) activity in adrenal gland and brain of the transgenic mice carrying an 11-kb fragment containing the entire human TH gene. Injection of 5 mg nicotine/kg (as free base) for 3 days caused a statistically significant increase in vitro TH activity in the adrenal gland, whereas brain TH activity was not affected at all. The adrenal gland of non-transgenic C57BL/6J mice treated in the same way as for transgenic mice tended to enhance TH activity, although not to a significant level. This observation might indicate the possibility that the machinery used by nicotine in regulating the properties or expression of TH in the adrenal gland should be similar between transgenic and non-transgenic mice.