Hypertension is a leading cause of human cardiovascular morbidity and mortality, with a prevalence rate of 25%-30% in the adult Caucasian population in the USA[155] and in Europe. The primary determinants of essential hypertension , which represents 95% of the hypertensive population[156], have not been elucidated despite intensive research. Studies of large populations, of twins, and of adoptive siblings, provide evidence for a strong genetic component in the regulation of blood pressure [157]. On the basis of these observations molecular determinants which contribute to the pathogenesis of hypertension have been searched for. There is evidence suggesting that 30% of the variation of blood pressure may be genetically determined[158].
Epidemiologic studies indicate a continuous distribution of blood pressure in the population, and the genetic disorder appears to be polygenic and therefore does not follow simple Mendelian patterns of inheritance[159]. It is likely that several genes interact with environmental stimuli to produce high blood pressure in susceptible persons[158].
It has been shown recently that the locus of the angiotensinogen (AGT) gene, which encodes a key component of the renin-angiotensin system, is linked to essential hypertension both in Caucasians and in African Caribbeans [10, 12]. This finding represents a major advance in the search for genes associated with essential hypertension. The exact mutations within the AGT gene influencing disease susceptibility remain to be discovered.
Among those environmental factors possibly contributing to the development
of essential hypertension salt intake appears to be of prominent
importance [160].
There is convincing evidence, that in populations
with a high salt intake the prevalence of essential hypertension is
increased [7].
The mechanisms by
which salt causes hypertension are not completely understood. Of particular
interest is the
interaction of the sympathetic nervous system and salt intake. The
sympathetic nervous system
plays a key role in the regulation of blood pressure. It acts via
neurotransmitters (epinephrine, nor-epinephrine) on adrenergic receptors.
The cellular equipment with various receptor subtypes
defines the cellular response. In vascular smooth muscle cells, the main
determinants of the
peripheral resistance and hence the blood pressure, vasoconstriction is
mediated by 
and
receptors and vasodilatation by 
receptors. It has been
shown recently, that the
blood pressure response to salt is at least in part explained by changes of
the densities of these
adrenoceptor subtypes: a high salt intake causes a rise of
adrenoceptors (and promotes therefore vasoconstriction) and a fall of
receptors (resulting in a blunted vasodilation)
[161]. A subpopulation of normotensives is especially prone to blood
pressure rise when exposed to a high salt intake (this group is coined
salt sensitive). On contrast,
a substantial proportion of the population (about 60-70%) are salt resistant,
i.e. they demonstrate no rise of blood pressure when consuming a high salt
diet. In longitudinal studies it has been
shown that salt sensitivity in normotensives may be a precursor state of
essential hypertension [162].
A genetic basis of salt sensitivity has been suggested recently when it
appeared that in cell culture skin fibroblasts from salt sensitive subjects
express less than half the number of adrenoceptors as compared to
salt resistant subjects [163]. This finding prompted us to study the
genetics of the adrenoceptor in greater detail.
The etiological heterogeneity and multifactorial determination that
characterize diseases as common as hypertension expose the limitations of the
classical genetic arsenal. Definition of phenotype, model of inheritance,
optimal familial structures, and candidate gene impose critical strategic
choices[164, 165]. Therefore the establishing of a direct
phenotype , the -adrenoceptor density, was tried for easier
interpretation of the results.
Almost 50 years after AHLQUIST[19] first
uncovered evidence of the heterogeneity of adrenergic receptors, the number of
receptor subtypes is still unclear, although nine subtypes are well documented
(three subtypes each of -, -, and 
-adrenergic
receptors). Each type preferentially links to members of a subfamily of G
proteins: to 
, to 
, and
to 
, and in turn to effector molecules to which those G
proteins link ( to phospholipase 
,
and to adenylyl cyclase). The number of activated G proteins
exceeds the number of corresponding receptors and effectors; thus, the
activation of G proteins amplifies signaling by adrenergic receptors. Receptor
desensitization , mediated in part by
G-protein-receptor kinases and
-arrestins , is involved in decreasing
the ability of agonists to activate adrenergic receptors. Alterations in
adrenergic receptors have a role in many clinical settings. Recent data suggest
a role for the increased expression of G-protein-receptor kinases in hearts of
patients with congestive heart failure and for genetic polymorphisms in
-adrenergic receptors
in patients with obesity. Studies using molecular and biochemical techniques
are likely to provide additional new and unexpected insights into the role of
adrenergic receptors in both normal physiologic function and disease.
Receptor biosynthesis, processing, and insertion in the plasma membrane are not well understood. The regulation of adrenergic receptors by receptor-specific agonist s and antagonist s has been actively studied for many years, whereas the mechanism by which a high salt diet influences adrenergic receptors is not known yet. Given the widespread expression of adrenergic receptors and their role in regulating a wide variety of events, it is conceivable that alterations in these receptors have been suspected in many clinical settings (see table tab:clin).
Table 5.1: Clinical Conditions associated with possible alterations in adrenergic
receptors
As salt sensitivity resembles a pre-hypertensive state, we believe that the
human adrenergic receptor gene should be considered as a candidate
gene involved
in the development of essential hypertension .
The aim of this thesis was to search for mutations of the
adrenoceptor in several subjects and to develop a fast screening method in
order to eventually
determine the frequencies of alleles of the adrenergic receptor gene
in the general population, in salt sensitives and salt resistants, and in
hypertensive
subjects. Recent measurements of -adreneoceptor mRNA content
in cultured human skin fibroblasts revealed no difference irrespective of the
number of 2ar expressed on the cell surface[15]. These findings are in
line with data obtained in fat cells [166] and point towards the
possibility of a modification of adrenoceptor density at a level
beyond gene expression.
Mutational analysis of the 2ar gene showed that
N-glycosylation at Asn 
and Asn 
is important for correct trafficking of the receptor molecule
through the cell. Glycosylation deficient mutants showed in COS-7 cells a 50%
decrease in the level of accumulation of adrenergic receptor on the
cell surface[57]. The G 
A codon change results in a
Gly 
Arg primary structure change. The Gly Arg
mutation resembles the X within the Asn X Ser/Thr
glycosylation consensus sequence. The influence of the Gly Arg
amino acid change on the glycosylation pattern of the adrenergic
receptor protein is not known.
The secondary structure of mRNA is known to influence translation efficacy.
Silent mutations may have a significant impact on secondary mRNA structure.
Whether the silent mutations described in this thesis are associated with
alterations of secondary mRNA structure, mRNA stability, or translation
efficiency remains to be clarified. The GCG program
mfold
was used
to model secondary mRNA structures obtained with two of the detected mutations
(position 1309 and 1786); this analysis revealed altered secondary mRNA
structures and energies.
Although the number of samples used in this study was low, the point mutations
at positions 1309 and 1786 show a tendency towards higher (1309) or
lower adrenoceptor densities (1786), respectively. This can be
further demonstrated by
plotting the actual adrenergic receptor density versus
genotypes
(see figure fig:1786dens). Due to the fact that this thesis resembles a pilot study only a small
number of subjects was involved. This limitation will be overcome in the very near future
by an international joint study with Mark CAULFIELD
(St. Bartholomew's Hospital, London) using DNA from several hundreds of
clinically well characterized subjects [10].
Table 5.2: Alleles of the human adrenergic receptor gene in relation to
adrenoceptor expression on cultured skin
fibroblasts
The fact that in this small association study no direct relationship
of adrenoceptor alleles and salt sensitivity or hypertension has
been shown does not preclude the view
that, on the basis of previous studies [163], the adrenergic
receptor should be considered as one of the candidate gene s for the
development of essential hypertension.
Clear answers to the question whether or not the adrenergic receptor
is involved in the pathogenesis of essential hypertension can be expected
from large scale linkage studies.
Genetic polymorphism is a conditio sine qua non for linkage studies. This thesis work has
provided evidence for a polymorphism of the human 2ar gene. The
six 2ar alleles described so far provide sufficiently polymorphic genetic
markers to embark on large scale family studies.
Figure 5.1: Adrenoceptor genotype at position 1786 in relation to receptor
density
Figure 5.2: Adrenoceptor genotype at position 1309 in relation to receptor
density